WO2024249812A2

WO2024249812A2 - Egf variants and methods of use thereof

Info

Publication number: WO2024249812A2
Application number: PCT/US2024/031938
Authority: WO
Inventors: Zachary CROOK; Natalie Winblade Nairn
Original assignee: Blaze Bioscience Inc
Current assignee: Blaze Bioscience Inc
Priority date: 2023-06-01
Filing date: 2024-05-31
Publication date: 2024-12-05
Anticipated expiration: 2025-12-01
Also published as: WO2024249812A3

Abstract

Described herein are compositions and methods for selective depletion of EGFR target molecules using a recyclable receptor-binding mediated complex to elicit uptake or endocytosis and cellular degradation of the target molecule. Exemplary compositions containing a peptide, such as a CDP peptide that binds a transferrin receptor, can be linked to a peptide that binds an EGFR target molecule. Such compositions can be used to selectively deplete EGFR from the cell surface or selectively recruit EGFR to endosomes via transferrin receptor-mediated endocytosis of the composition and the bound target molecule. Once inside the endosome, the acidic pH can lead to release of the EGFR from the composition due to pH-dependent binding of the composition for the target molecule, and the transferrin receptor portion is recycled back to the cell surface for "reloading". The EGFR can then be trafficked into lysosomes wherein it is degraded.

Description

EGF VARIANTS AND METHODS OF USE THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/470,404, entitled “EGF VARIANTS AND METHODS OF USE THEREOF,” filed June 1, 2023, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 22, 2024, is named “438542- 725021_SL. xml” and is 757,899 bytes in size.

BACKGROUND

[0003] Accumulation or over-expression of soluble and cell surface proteins is indicated in a variety of human diseases, ranging from neurodegenerative diseases to cancer. Furthermore, numerous diseases are associated with mutations in soluble or cell surface proteins resulting in constitutive activity, resistance to treatment, or dominant negative activity. However, many of these proteins have been deemed “undruggable,” “difficult to drug,” or “yet to be drugged” targets due to challenges in targeting them with small molecule therapeutics. For example, the oncoprotein EGFR drives cell growth through both scaffolding and kinase functions, and current therapeutic modalities typically address one but not the other, leaving cancer cells prone to continued signaling through mutation or other adaptations. There is a need for compositions and methods to target and selectively deplete soluble and cell surface proteins associated with disease. In addition, the oncoprotein EGFR drives cell growth through both scaffolding and kinase functions, and current therapeutic modalities typically address one but not the other, leaving cancer cells prone to continued signaling through mutation or other adaptations.

Elimination of the entire EGFR protein would simultaneously disrupt both of its growth-driving functions. There is therefore a need for compositions and methods to target and selectively deplete EGFR associated with cancer, which can also be accomplished by engineering of EGF itself.

SUMMARY

[0004] In various aspects, the present disclosure provides an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R.

[0005] In some aspects, the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof. In some aspects, the sequence further comprises one or more mutations relative to SEQ ID NO: 317 comprising DI 1R, I23S, V35E, E51P, L52E, R53E, or a combination thereof. In some aspects, the sequence further comprises one or more mutations relative to SEQ ID NO: 317 comprising E51H, L52H, R53H, or a combination thereof. In some aspects, the EGFR-binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 458 - SEQ ID NO: 493 or a fragment thereof. In some aspects, the sequence comprises SEQ ID NO: 494. In some aspects, the sequence comprises any one of SEQ ID NO: 458 - SEQ ID NO: 493. In some aspects, the fragment comprises at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues.

[0006] In some aspects, the EGFR-binding peptide is capable of binding to EGFR without activating the EGFR. In some aspects, the EGFR-binding peptide blocks binding of EGF to EGFR when the EGFR-binding peptide is bound to the EGFR. In some aspects, the EGFR- binding peptide inhibits EGFR when the EGFR-binding peptide is bound to the EGFR. In some aspects, the EGFR-binding peptide prevents dimerization of EGFR when the EGFR-binding peptide is bound to the EGFR.

[0007] In various aspects, the present disclosure provides a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR-binding peptide has affinity for a target molecule.

[0008] In some aspects, the EGFR-binding peptide is the EGFR-binding peptide as described herein. In some aspects, the affinity of the EGFR-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH- independent. In some aspects, the affinity of the EGFR-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH dependent. In some aspects, the affinity of the EGFR-binding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for the cellular receptor, or both is ionic strength dependent.

[0009] In some aspects, the cellular receptor-binding peptide is a transferrin receptor-binding peptide or a PD-L1 -binding peptide. In some aspects, the cellular receptor is a transferrin receptor or PD-L1. In some aspects, the cellular receptor is a cation-independent mannose 6 phosphate receptor (CI-M6PR), an asialoglycoprotein receptor (ASGPR), CXCR7, folate receptor, or Fc receptor (including but not limited to neonatal Fc receptor (FcRn) or FcyRIIb). [0010] In some aspects, the cellular receptor-binding peptide binds to the cellular receptor at a pH of from pH 4.5 to pH 7.4, from pH 5.5 to pH 7.4, from pH 5.8 to pH 7.4, or from pH 6.5 to pH 7.4. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with a dissociation rate constant (k_off or kd) of no more than 1 s'¹, no more than 5x10'¹ s'¹, no more than 2x10'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s'¹, or no more than 2x1 O'⁴ s'¹ at pH 5.5.

[0011] In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is pH-independent. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is pH-dependent. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is pH-independent. In some aspects, the affinity of the cellular receptorbinding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5 -fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the dissociation rate constant (koff or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the dissociation rate constant (koff or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0012] In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is pH dependent. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor decreases as pH decreases. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.5. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.8.

[0013] In some aspects, the affinity of the EGFR-binding peptide for the target molecule is pH dependent. In some aspects, the affinity of the EGFR-binding peptide for the target molecule decreases as pH decreases. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is higher at a higher pH than at a lower pH. In some aspects, the higher pH is pH 7.4, pH 7.2, pH 7.0, or pH 6.8. In some aspects, the lower pH is pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is higher at pH 7.4 than at pH 6.0. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is higher at pH 7.4 than at pH 5.5. In some aspects, the affinity of the EGFR-binding peptide for the target molecule is higher at pH 7.4 than at pH 5.8.

[0014] In some aspects, the EGFR-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no more than 500 nM, no more than 200 nM, 100 nM, no more than 50 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 1 nM, or no more than 0.1 nM at pH 7.4.

[0015] In some aspects, the EGFR-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1x10'¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5xl0’⁵ s’¹, or no more than 2xl0’⁵ s’¹ at pH 7.4. In some aspects, the EGFR-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.5. In some aspects, the EGFR-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x10’ ² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.8.

[0016] In some aspects, the dissociation rate constant (koff or kd) for EGFR-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.5 than at pH 7.4. In some aspects, the dissociation rate constant (koff or kd) for EGFR-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.8 than at pH 7.4.

[0017] In some aspects, the EGFR-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.5. In some aspects, the EGFR-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.8.

[0018] In some aspects, the affinity of the EGFR-binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.5. In some aspects, the affinity of the EGFR-binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.8.

[0019] In some aspects, the affinity of the EGFR-binding peptide for the target molecule at pH 7.4 is less than 0.5-fold, less than 1-fold, less than, 1.5-fold, less than 2-fold, less than 3-fold, or less than 10-fold, greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.8.

[0020] In some aspects, the EGFR-binding peptide comprises one or more histidine amino acid residues. In some aspects, the affinity of the EGFR-binding peptide for the target molecule decreases as ionic strength increases. In some aspects, the EGFR-binding peptide comprises one or more polar or charged amino acid residues capable of forming polar or charge-charge interactions with the target molecule. [0021] In some aspects, the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the EGFR-binding peptide. In some aspects, the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the EGFR-binding peptide via a polymer linker. In some aspects, the polymer linker is a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer comprising proline, alanine, serine, or a combination thereof, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, a palmitic acid, an albumin, or an albumin binding molecule. [0022] In some aspects, the cellular receptor-binding peptide and the EGFR-binding peptide form a single polypeptide chain. In some aspects, the peptide complex comprises a dimer dimerized via a dimerization domain. In some aspects, a distance between the cellular receptorbinding peptide and the EGFR-binding peptide is at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, or at least 100 nm. In some aspects, the dimerization domain comprises an Fc domain. In some aspects, the dimer is a homodimer dimerized via a homodimerization domain. In some aspects, the homodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 535, SEQ ID NO: 706, or SEQ ID NO: 246. In some aspects, the dimer is a heterodimer dimerized via a first heterodimerization domain and a second heterodimerization domain. In some aspects, the first heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 536, SEQ ID NO: 707, or SEQ ID NO: 709. In some aspects, the second hetero dimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 537, SEQ ID NO: 708, or SEQ ID NO: 710.

[0023] In some aspects, the EGFR-binding peptide is linked to the dimerization domain via a peptide linker. In some aspects, the cellular receptor-binding peptide is linked to the dimerization domain via a peptide linker. In some aspects, the cellular receptor-binding peptide is linked to the EGFR-binding peptide via a peptide linker. In some aspects, the peptide linker has a length of from 1 to 50 amino acid residues, from 2 to 40 amino acid residues, from 3 to 20 amino acid residues, or from 3 to 10 amino acid residues. In some aspects, the peptide linker comprises glycine and serine amino acids. In some aspects, the peptide linker has a persistence length of no more than 6 A, no more than 8 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, no more than 50 A, no more than 75 A, no more than 100 A, no more than 150 A, no more than 200 A, no more than 250 A, or no more than 300 A. In some aspects, the peptide linker is derived from an immunoglobulin peptide. In some aspects, the peptide linker is derived from a doubleknot toxin peptide. In some aspects, the peptide linker comprises a sequence of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 223 - SEQ ID NO: 223 - SEQ ID NO: 227, SEQ ID NO: 194, SEQ ID NO: 391, SEQ ID NO: 538, or SEQ ID NO: 540 - SEQ ID NO: 541.

[0024] In some aspects, the cellular receptor-binding peptide, the EGFR-binding peptide, peptide complex, or a combination thereof comprises a miniprotein, a nanobody, an antibody, an antibody fragment, an scFv, a DARPin, or an affibody. In some aspects, the antibody comprises an IgG, or wherein the antibody fragment comprises a Fab, a F(ab)2, an scFv, or an (scFv)2. In some aspects, the miniprotein comprises a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some aspects, the cellular receptor-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the EGFR-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the peptide complex comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the cellular receptorbinding peptide comprises at least six cysteine residues. In some aspects, the at least six cysteine residues are positioned at amino acid positions 4, 8, 18, 32, 42, and 46 of the cellular receptorbinding peptide. In some aspects, the at least six cysteine residues form at least three disulfide bonds.

[0025] In some aspects, the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 148 - SEQ ID NO: 177. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64.

[0026] In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 96. In some aspects, the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 96.

[0027] In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 66, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 66. In some aspects, the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 66.

[0028] In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 65, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 65. In some aspects, the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 65.

[0029] In some aspects, the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 392 - SEQ ID NO: 399. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241.

[0030] In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 187, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 400, or SEQ ID NO: 401 or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 187. In some aspects, the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 187.

[0031] In some aspects, the fragment comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues. [0032] In some aspects, the cellular receptor-binding peptide comprises one or more histidine residues at a cellular receptor-binding interface. In some aspects, the EGFR-binding peptide comprises one or more histidine residues at a EGFR-binding interface. In some aspects, the target molecule comprises an EGFR. In some aspects, the EGFR is wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, a panitumumab-resistant EGFR, or a combination thereof. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0033] In some aspects, an off rate of the cellular receptor-binding peptide from the cellular receptor is slower than a recycling rate of the cellular receptor. In some aspects, a half-life of dissociation of the cellular receptor-binding peptide from the cellular receptor is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, no faster than 20 minutes, no faster than 30 minutes, no faster than 45 minutes, no faster than 60 minutes, no faster than 90 minutes, or no faster than 120 minutes. In some aspects, a rate of dissociation of the EGFR-binding peptide from the target molecule is faster than a recycling rate of the cellular receptor. In some aspects, a half-life of dissociation of the target binding-binding peptide from the target molecule is less than 10 seconds, less than 20 seconds, less than 30 seconds, less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, or less than 60 minutes in endosomal conditions.

[0034] In some aspects, the peptide complex is capable of being endocytosed via receptor- mediated endocytosis. In some aspects, the receptor-mediated endocytosis is transferrin receptor-mediated endocytosis. In some aspects, the receptor-mediated endocytosis is PD-L1- mediated endocytosis. In some aspects, the cellular receptor-binding peptide remains bound to the cellular receptor inside an endocytic vesicle. In some aspects, the peptide complex is recycled to the cell surface when the cellular receptor-binding peptide is bound to the cellular receptor and the cellular receptor is recycled. In some aspects, the target molecule is released or dissociated from the EGFR-binding peptide after the peptide complex is endocytosed via receptor-mediated endocytosis.

[0035] In some aspects, the target molecule is an extracellular protein, a circulating protein, or a soluble protein. In some aspects, the target molecule is a cell surface protein. In some aspects, the target molecule is a transmembrane protein.

[0036] In some aspects, the peptide complex further comprises a half-life modifying agent coupled to the cellular receptor-binding peptide, the EGFR-binding peptide, or both. In some aspects, the half-life modifying agent is a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin. In some aspects, the molecule that binds to albumin is a serum albumin-binding peptide. In some aspects, the serum albumin-binding peptide comprises a sequence of any one of SEQ ID NO: 178, SEQ ID NO: 179, or SEQ ID NO: 193.

[0037] In some aspects, the cellular receptor-binding peptide, the EGFR-binding peptide, or both is recombinantly expressed. In some aspects, the EGFR-binding peptide is configured to dissociate from the target molecule at pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. In some aspects, the cellular receptor-binding peptide is configured to dissociate from the cellular receptor at pH 6.5, pH 6.0, pH 5.5, pH 5.0, or pH 4.5.

[0038] In some aspects, the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 507, SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526. In some aspects, the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 503, or SEQ ID NO: 506.

[0039] In various aspects, the present disclosure provides a peptide-active agent complex comprising a peptide complexed to an active agent, wherein the peptide comprises the EGFR- binding peptide as described herein or the peptide complex as described herein.

[0040] In some aspects, the active agent comprises a peptide, a peptidomimetic, an oligonucleotide, a DNA, an RNA, an antibody, a single chain variable fragment (scFv), an antibody fragment, an aptamer, or a small molecule. In some aspects, the DNA comprises cDNA, ssDNA, or dsDNA. In some aspects, the RNA comprises RNAi, microRNA, snRNA, dsRNA, or an antisense oligonucleotide. In some aspects, the active agent is a therapeutic agent or a detectable agent. In some aspects, the detectable agent comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, a radioisotope, nanopartide, a paramagnetic metal ion, or a combination thereof. In some aspects, the therapeutic agent comprises a chemical agent, a small molecule, a therapeutic, a drug, a peptide, an antibody protein, any fragment thereof, or any combination thereof.

[0041] In some aspects, the therapeutic agent comprises an oncology agent, an autoimmune disease agent, an acute and chronic neurodegeneration agent, a pain management agent, or an anti-cancer agent. In some aspects, the anti-cancer agent comprises a radionuclide, radioisotope, a chemotherapeutic agent, a platinum therapeutic, a toxin, an enzyme, a sensitizing drug, an anti- angiogenic agent, cisplatin, an anti-metabolite, an anti-metabolic therapeutic, a mitotic inhibitor, a growth factor inhibitor, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, or amifostine. In some aspects, the anti-cancer agent targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells.

[0042] In various aspects, the present disclosure provides a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent.

[0043] In some aspects, the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof. In some aspects, the EGFR-binding peptide is the EGFR-binding peptide as described herein.

[0044] In various aspects, the present disclosure provides a pharmaceutical composition comprising: a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent.

[0045] In some aspects, the peptide complex is the peptide complex as described herein or the peptide-active agent complex as described herein.

[0046] In various aspects, the present disclosure provides a method of administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent, to a subject.

[0047] In some aspects, the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof. In some aspects, the EGFR-binding peptide is the EGFR-binding peptide as described herein.

[0048] In various aspects, the present disclosure provides a method of administering a pharmaceutical composition comprising: a peptide complex comprising: (i) a cellular receptorbinding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR- binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent, to a subject.

[0049] In some aspects, the peptide complex is the peptide complex as described herein or the peptide-active agent complex as described herein. In some aspects, the pharmaceutical composition is the pharmaceutical composition as described herein.

[0050] In various aspects, the present disclosure provides a method of inhibiting EGFR in a subject, the method comprising: administering to the subject a pharmaceutical composition, wherein the pharmaceutical composition comprises: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR.

[0051] In some aspects, the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof. In some aspects, the EGFR-binding peptide is the EGFR-binding peptide as described herein. [0052] In various aspects, the present disclosure provides a method of inhibiting EGFR in a subject, the method comprising: administering to the subject a pharmaceutical composition, wherein the pharmaceutical composition comprises: a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR- binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR.

[0053] In some aspects, the peptide complex is the peptide complex as described herein or the peptide-active agent complex as described herein. In some aspects, the pharmaceutical composition is the pharmaceutical composition as described herein. In some aspects, the EGFR- binding peptide inhibits activation of the EGFR by disrupting multimerization, dimerization, or heterodimerization of the EGFR on the cell of the subject that expresses EGFR.

[0054] In various aspects, the present disclosure provides a method of selectively depleting a target molecule, the method comprising: (a) contacting a peptide complex to a cell expressing a cellular receptor, wherein the peptide complex comprises: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR- binding peptide has affinity for a target molecule; (b) binding the EGFR-binding peptide to the target molecule under extracellular conditions; (c) binding the cellular receptor-binding peptide to the cellular receptor under extracellular conditions; and (d) endocytosing the peptide complex, the target molecule, and the cellular receptor into an endocytic or lysosomal compartment, thereby depleting the target molecule.

[0055] In some aspects, the peptide complex as described herein or the peptide-active agent complex as described herein. In some aspects, the method further comprises (e) dissociating the EGFR-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both, under endosomal or lysosomal conditions. In some aspects, the method further comprises (f) degrading the target molecule, thereby further depleting the target molecule. In some aspects, the method further comprises recycling the peptide complex and the cellular receptor to the cell surface.

[0056] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent, to the subject, thereby treating the disease or condition.

[0057] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising: administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition.

[0058] In some aspects, the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof. In some aspects, the EGFR-binding peptide is the EGFR-binding peptide as described herein.

[0059] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising: a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR- binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent, to the subject, thereby treating the disease or condition.

[0060] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising: administering a pharmaceutical composition comprising: a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR- binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-b inding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition.

[0061] In some aspects, the peptide complex as described herein or the peptide-active agent complex as described herein. In some aspects, the pharmaceutical composition is the pharmaceutical composition as described herein.

[0062] In some aspects, the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, or a panitumumab- resistant EGFR. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0063] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising: (a) administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises: a peptide complex comprising: (i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent; (b) binding the EGFR-binding peptide under extracellular conditions to a target molecule associated with the disease or condition on a cell of the subject expressing the target molecule and a cellular receptor; (c) binding the cellular receptor-binding peptide under extracellular conditions to the cellular receptor on the cell of the subject; and (d) endocytosing the peptide complex, the target molecule, and the cellular receptor, thereby treating the disease or condition.

[0064] In some aspects, the peptide complex is the peptide complex as described herein or the peptide-active agent complex as described herein. In some aspects, the pharmaceutical composition is the pharmaceutical composition as described herein.

[0065] In some aspects, the method further comprises (e) dissociating the EGFR-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both under endosomal conditions. In some aspects, the method further comprises (f) degrading the target molecule.

[0066] In some aspects, the target molecule comprises an EGFR. In some aspects, the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, or a panitumumab-resistant EGFR. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0067] In some aspects, the disease or condition is a cancer. In some aspects, the cancer expresses EGFR, overexpresses EGFR, or contains mutant EGFR. In some aspects, the cancer is breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, blood-cell-derived cancer, lung cancer, sarcoma, stomach cancer, a gastrointestinal cancer, glioblastoma, head and neck cancer, squamous head and neck cancer, non-small-cell lung cancer, squamous non-small cell lung cancer, pancreatic cancer, ovarian cancer, endometrial cancer, blood cancer, skin cancer, liver cancer, kidney cancer, or colorectal cancer. In some aspects, the cancer is TKI-resistant, cetuximab-resistant, necitumumab-resistant, or panitumumab-resistant. In some aspects, the cancer has one or more of the following: overexpresses EGFR, KRAS mutation, KRAS G12S mutation, KRAS G12C mutation, PTEN loss, EGFR exonl9 deletion, EGFR L858R mutation, EGFR T790M mutation, PIK3CA mutation, TP53 R273H mutation, PIK3CA amplification, PIK3CA G118D, TP53 R273H, EGFR C797X mutation, EGFR G724S mutation, EGFR L718Q mutation, EGFR S768I mutation, an EGFR mutation, a cetuximab-resistant EGFR, a panitumumab-resistant EGFR, or a combination thereof.

[0068] In some aspects, the cancer expresses or has upregulated c-MET, Her2, Her3 that heterodimerizes with EGFR. In some aspects, the cancer is a primary cancer, an advanced cancer, a metastatic cancer, a metastatic cancer in the central nervous system, a primary cancer in the central nervous system, metastatic colorectal cancer, metastatic head and neck cancer, metastatic non-small-cell lung cancer, metastatic breast cancer, metastatic skin cancer, a refractory cancer, a KRAS wild type cancer, a KRAS mutant cancer, or an exon20 mutant non- small-cell lung cancer.

[0069] In some aspects, the method further comprises administering an additional therapy to the subject. In some aspects, the additional therapy is adjuvant, first-line, or combination therapy. In some aspects, the additional therapy targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. In some aspects, targeting of other oncogenic signaling pathways comprises administration of inhibitors of MEK/ERK pathway signaling, PI3K/AKT pathway signaling, JAK/STAT pathway signaling, or WNT/p-catenin pathway signaling. In some aspects, targeting of immune response pathways comprises PD-1/PD-L1 checkpoint inhibition. In some aspects, directly driving an immune response to cancer cells comprises bispecific T cell engagers or chimeric antigen receptor expressing T cells. In some aspects, targeting disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells comprises administering seno lytic agents to a subject. [0070] In some aspects, the additional therapy comprises administering fluorouracil, FOLFIRI, irinotecan, FOLFOX, gemcitabine, or cisplatin, irinotecan, oxiplatin, fluoropyrimidine to the subject.

[0071] In some aspects, the method further comprises forming a ternary complex between the peptide complex, the target molecule, and the cellular receptor. In some aspects, formation of the ternary complex increases, facilitates, or stabilizes recycling or turnover of the cellular receptor, the target molecule, or both. In some aspects, formation of the ternary complex increases, facilitates, or stabilizes binding of the target molecule to the cellular receptor. In some aspects, the peptide complex binds at higher levels to cells that overexpress the target molecule and the cellular receptor than to cells that have lower levels of the target molecule or the cellular receptor or both.

[0072] In some aspects, the peptide complex has a larger, longer, or wider therapeutic window as compared to an alternative therapy. In some aspects, the alternative therapy is not recycled to the cell surface. In some aspects, the alternative therapy is a lysosomal targeting therapy, a ubiquitin-proteosome system (UPS) targeting therapy, a non-selective therapeutic agent, an existing biologic, or a lysosomal delivery molecule. In some aspects, the peptide complex or the EGFR-binding peptide is administered at lower molar dosage than alternative therapies. In some aspects, the peptide complex or the EGFR-binding peptide binds at higher levels to cancer cells than to normal cells.

[0073] In some aspects, the peptide complex or the EGFR-binding peptide has a higher antiproliferative effect, a higher target molecule depletion effect, or a higher viability effect on cancer cells than on normal cells in vitro or in vivo. In some aspects, the peptide complex or the EGFR-binding peptide has a larger, longer, or wider therapeutic window than an anti-EGFR antibody or a TKI. In some aspects, the peptide complex or the EGFR-binding peptide has lower toxicity on skin or on keratinocytes than an anti-EGFR antibody or a TKI.

[0074] In some aspects, the method further comprises causing remission in, reducing, ameliorating, or ablating the disease or condition. In some aspects, the method further comprises causing remission in, reducing, ameliorating, or ablating the cancer.

[0075] In various aspects, the present disclosure provides an EGFR-binding peptide comprising a sequence having at least 70% sequence identity to SEQ ID NO: 317 and comprising at least one mutation relative to SEQ ID NO: 317.

[0076] In some aspects, the EGFR-binding peptide comprises a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue; wherein the EGFR-binding peptide comprises: seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue. [0077] In some aspects, the first cysteine amino acid residue is at position 6 of the EGFR- binding peptide, the second cysteine amino acid residue is at position 14 of the EGFR-binding peptide, the third cysteine amino acid residue is at position 20 of the EGFR-binding peptide, the fourth cysteine amino acid residue is at position 31 of the EGFR-binding peptide, the fifth cysteine amino acid residue is at position 33 of the EGFR-binding peptide, and the sixth cysteine amino acid residue is at position 42 of the EGFR-binding peptide.

[0078] In some aspects, the at least one mutation comprises an amino acid substitution of DI 1R, I23S, V35E, S51P, L52E, R53E, M21R, A30W, I38D, W49R, V34S, Q43I, Q43V, Q43W, Q43Y, K48N, K48T, K48A, K48L, E51S, E51H, L52H, R53H, or a combination thereof. In some aspects, the at least one mutation comprises an amino acid substitution of M21R, A30W, 138D, W49R, or a combination thereof. In some aspects, the at least one mutation comprises an amino acid substitution of DI 1R, I23S, V35E, S51P, L52E, R53E, or a combination thereof. In some aspects, the at least one mutation comprises an amino acid substitution of E51H, L52H, R53H, or a combination thereof.

[0079] In various aspects, the present disclosure provides an EGFR-binding peptide comprising a sequence of SEQ ID NO: 314.

[0080] In various aspects, the present disclosure provides a n EGFR-binding peptide comprising a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

[0081] In some aspects, the EGFR-binding peptide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. In some aspects, the EGFR-binding peptide is capable of binding to EGFR without activating the EGFR. In some aspects, the EGFR-binding peptide blocks binding of EGF to EGFR when the EGFR-binding peptide is bound to the EGFR. In some aspects, the EGFR-binding peptide inhibits EGFR when the EGFR-binding peptide is bound to the EGFR. In some aspects, the EGFR-binding peptide prevents dimerization of EGFR when the EGFR- binding peptide is bound to the EGFR.

[0082] In various aspects, the present disclosure provides a peptide complex comprising: a) a cellular receptor-binding peptide; and b) a target-binding peptide complexed with the cellular receptor-binding peptide, wherein the target-binding peptide has affinity for a target molecule, and wherein the target-binding peptide comprises the EGFR-binding peptide as described herein. [0083] In various aspects, the present disclosure provides a peptide complex comprising: a) a cellular receptor-binding peptide; and b) a target-binding peptide complexed with the cellular receptor-binding peptide, wherein the target-binding peptide has affinity for a target molecule, and wherein the target-binding peptide comprises a sequence of SEQ ID NO: 314 or a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

[0084] In some aspects, the affinity of the target-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH- independent. In some aspects, the affinity of the target-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH dependent. In some aspects, the affinity of the target-binding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for the cellular receptor, or both is ionic strength dependent.

[0085] In some aspects, the target binding peptide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. In some aspects, the target binding peptide comprises a sequence of any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

[0086] In some aspects, the cellular receptor-binding peptide is a transferrin receptor-binding peptide or a PD-L1 -binding peptide. In some aspects, the cellular receptor is a transferrin receptor or PD-L1. In some aspects, the cellular receptor is a cation-independent mannose 6 phosphate receptor (CI-M6PR), an asialoglycoprotein receptor (ASGPR), CXCR7, folate receptor, or Fc receptor (including but not limited to neonatal Fc receptor (FcRn) or FcyRIIb). [0087] In some aspects, the cellular receptor-binding peptide binds to the cellular receptor at a pH of from pH 4.5 to pH 7.4, from pH 5.5 to pH 7.4, from pH 5.8 to pH 7.4, or from pH 6.5 to pH 7.4. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. In some aspects, the cellular receptor-binding peptide is capable of binding the cellular receptor with a dissociation rate constant (k_off or kd) of no more than 1 s'¹, no more than 5x1 O'¹ s'¹, no more than 2x1 O'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s'¹, or no more than 2x1 O'⁴ s'¹ at pH 5.5.

[0088] In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is pH-independent. In some aspects, the affinity of the target-binding peptide for the target molecule is pH-dependent. In some aspects, the affinity of the target-binding peptide for the target molecule is pH-independent.

[0089] In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the dissociation rate constant (koff or kd) of the cellular receptorbinding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some aspects, the dissociation rate constant (koff or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0090] In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is pH dependent. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor decreases as pH decreases. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.5. In some aspects, the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.8. In some aspects, the affinity of the target-binding peptide for the target molecule is pH dependent. In some aspects, the affinity of the target-binding peptide for the target molecule decreases as pH decreases. In some aspects, the affinity of the target-binding peptide for the target molecule is higher at a higher pH than at a lower pH. In some aspects, the higher pH is pH 7.4, pH 7.2, pH 7.0, or pH 6.8. In some aspects, the lower pH is pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. In some aspects, the affinity of the target-binding peptide for the target molecule is higher at pH 7.4 than at pH 6.0. In some aspects, the affinity of the targetbinding peptide for the target molecule is higher at pH 7.4 than at pH 5.5. In some aspects, the affinity of the target-binding peptide for the target molecule is higher at pH 7.4 than at pH 5.8. [0091] In some aspects, the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no more than 500 nM, no more than 200 nM, 100 nM, no more than 50 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 1 nM, or no more than 0.1 nM at pH 7.4. In some aspects, the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1x10'¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5x10’⁵ s’¹, or no more than 2x10’⁵ s’¹ at pH 7.4.

[0092] In some aspects, the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.5. In some aspects, the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.8.

[0093] In some aspects, the dissociation rate constant (koff or kd) for target-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.5 than at pH 7.4. In some aspects, the dissociation rate constant (koff or kd) for target-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.8 than at pH 7.4.

[0094] In some aspects, the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.5. In some aspects, the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.8.

[0095] In some aspects, the affinity of the target-binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the target-binding peptide for the target molecule at pH 5.5. In some aspects, the affinity of the target-binding peptide for the target molecule at pH 7.4 is at least 1.5- fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the target-binding peptide for the target molecule at pH 5.8. In some aspects, the affinity of the target-binding peptide for the target molecule at pH 7.4 is less than 0.5-fold, less than 1-fold, less than, 1.5-fold, less than 2-fold, less than 3-fold, or less than 10-fold, greater than the affinity of the target-binding peptide for the target molecule at pH 5.8.

[0096] In some aspects, the target-binding peptide comprises one or more histidine amino acid residues. In some aspects, the affinity of the target-binding peptide for the target molecule decreases as ionic strength increases. In some aspects, the target-binding peptide comprises one or more polar or charged amino acid residues capable of forming polar or charge-charge interactions with the target molecule.

[0097] In some aspects, the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the target-binding peptide. In some aspects, the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the target-binding peptide via a polymer linker. In some aspects, the polymer linker is a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer comprising proline, alanine, serine, or a combination thereof, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, a palmitic acid, an albumin, or an albumin binding molecule. [0098] In some aspects, the cellular receptor-binding peptide and the target-binding peptide form a single polypeptide chain. In some aspects, the peptide complex comprises a dimer dimerized via a dimerization domain. In some aspects, the distance between the cellular receptor-binding peptide and the target-binding peptide is at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, or at least 100 nm. In some aspects, the dimerization domain comprises an Fc domain. In some aspects, the dimer is a homodimer dimerized via a homodimerization domain. In some aspects, the homodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 535, SEQ ID NO: 706, or SEQ ID NO: 246. In some aspects, the dimer is a heterodimer dimerized via a first hetero dimerization domain and a second heterodimerization domain. In some aspects, the first heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 536, SEQ ID NO: 707, or SEQ ID NO: 709. In some aspects, the second heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 537, SEQ ID NO: 708, or SEQ ID NO: 710.

[0099] In some aspects, the target-binding peptide is linked to the dimerization domain via a peptide linker. In some aspects, the cellular receptor-binding peptide is linked to the dimerization domain via a peptide linker. In some aspects, the cellular receptor-binding peptide is linked to the target-binding peptide via a peptide linker. In some aspects, the peptide linker has a length of from 1 to 50 amino acid residues, from 2 to 40 amino acid residues, from 3 to 20 amino acid residues, or from 3 to 10 amino acid residues. In some aspects, the peptide linker comprises glycine and serine amino acids. In some aspects, the peptide linker has a persistence length of no more than 6 A, no more than 8 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, no more than 50 A, no more than 75 A, no more than 100 A, no more than 150 A, no more than 200 A, no more than 250 A, or no more than 300 A. In some aspects, the peptide linker is derived from an immunoglobulin peptide. In some aspects, the peptide linker is derived from a double- knot toxin peptide. In some aspects, the peptide linker comprises a sequence of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 223 - SEQ ID NO: 223 - SEQ ID NO: 227, SEQ ID NO: 194, SEQ ID NO: 391, SEQ ID NO: 538, or SEQ ID NO: 540 - SEQ ID NO: 541.

[0100] In some aspects, the cellular receptor-binding peptide, the target-binding peptide, peptide complex, or a combination thereof comprises a miniprotein, a nanobody, an antibody, an antibody fragment, an scFv, a DARPin, or an affibody. In some aspects, the antibody comprises an IgG, or wherein the antibody fragment comprises a Fab, a F(ab)2, an scFv, or an (scFv)2. In some aspects, the miniprotein comprises a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some aspects, the cellular receptor-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the target-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the peptide complex comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. In some aspects, the cellular receptorbinding peptide comprises at least six cysteine residues. In some aspects, the at least six cysteine residues are positioned at amino acid positions 4, 8, 18, 32, 42, and 46 of the cellular receptorbinding peptide. In some aspects, the at least six cysteine residues form at least three disulfide bonds.

[0101] In some aspects, the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 148 - SEQ ID NO: 177. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 96. In some aspects, the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 96.

[0102] In some aspects, the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 392 - SEQ ID NO: 399. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241. In some aspects, the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 187, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 400, or SEQ ID NO: 401 or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 187. In some aspects, the cellular receptorbinding peptide comprises a sequence of SEQ ID NO: 187.

[0103] In some aspects, the fragment comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues. In some aspects, the cellular receptor-binding peptide comprises one or more histidine residues at a cellular receptor-binding interface. In some aspects, the target-binding peptide comprises one or more histidine residues at a target-binding interface.

[0104] In some aspects, the target-binding peptide is an EGFR-binding peptide. In some aspects, the target molecule comprises an EGFR. In some aspects, the EGFR is wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, or a combination thereof. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0105] In some aspects, an off rate of the cellular receptor-binding peptide from the cellular receptor is slower than a recycling rate of the cellular receptor. In some aspects, a half-life of dissociation of the cellular receptor-binding peptide from the cellular receptor is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, no faster than 20 minutes, no faster than 30 minutes, no faster than 45 minutes, no faster than 60 minutes, no faster than 90 minutes, or no faster than 120 minutes. In some aspects, a rate of dissociation of the target-binding peptide from the target molecule is faster than a recycling rate of the cellular receptor. In some aspects, a half-life of dissociation of the target binding-binding peptide from the target molecule is less than 10 seconds, less than 20 seconds, less than 30 seconds, less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, or less than 60 minutes in endosomal conditions.

[0106] In some aspects, the peptide complex is capable of being endocytosed via receptor- mediated endocytosis. In some aspects, the receptor-mediated endocytosis is transferrin receptor-mediated endocytosis. In some aspects, the receptor-mediated endocytosis is PD-L1- mediated endocytosis. In some aspects, the cellular receptor-binding peptide remains bound to the cellular receptor inside an endocytic vesicle. In some aspects, the peptide complex is recycled to the cell surface when the cellular receptor-binding peptide is bound to the cellular receptor and the cellular receptor is recycled. In some aspects, the target molecule is released or dissociated from the target-binding peptide after the peptide complex is endocytosed via receptor-mediated endocytosis.

[0107] In some aspects, the target molecule is an extracellular protein, a circulating protein, or a soluble protein. In some aspects, the target molecule is a cell surface protein. In some aspects, the target molecule is a transmembrane protein.

[0108] In some aspects, the peptide complex further comprises a half-life modifying agent coupled to the cellular receptor-binding peptide, the target-binding peptide, or both. In some aspects, the half-life modifying agent is a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin. In some aspects, the molecule that binds to albumin is a serum albumin-binding peptide. In some aspects, the serum albumin-binding peptide comprises a sequence of any one of SEQ ID NO: 178, SEQ ID NO: 179, or SEQ ID NO: 193.

[0109] In some aspects, the cellular receptor-binding peptide, the target-binding peptide, or both is recombinantly expressed. In some aspects, the target-binding peptide is configured to dissociate from the target molecule at pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. In some aspects, the cellular receptor-binding peptide is configured to dissociate from the cellular receptor at pH 6.5, pH 6.0, pH 5.5, pH 5.0, or pH 4.5.

[0110] In some aspects, the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 507, SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526. In some aspects, the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 503, or SEQ ID NO: 506.

[0111] In various aspects, the present disclosure provides a peptide-active agent complex comprising a peptide complexed to an active agent, wherein the peptide comprises the EGFR- binding peptide as described herein or the peptide complex as described herein.

[0112] In some aspects, the active agent comprises a peptide, a peptidomimetic, an oligonucleotide, a DNA, an RNA, an antibody, a single chain variable fragment (scFv), an antibody fragment, an aptamer, or a small molecule. In some aspects, the DNA comprises cDNA, ssDNA, or dsDNA. In some aspects, the RNA comprises RNAi, microRNA, snRNA, dsRNA, or an antisense oligonucleotide. In some aspects, the active agent is a therapeutic agent or a detectable agent. In some aspects, the detectable agent comprises a dye, a fhiorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, a radioisotope, nanopartide, a paramagnetic metal ion, or a combination thereof. In some aspects, the therapeutic agent is an anti-cancer agent. In some aspects, the anti-cancer agent comprises a radionuclide, radioisotope, a chemotherapeutic agent, a platinum therapeutic, a toxin, an enzyme, a sensitizing drug, an anti-angiogenic agent, cisplatin, an anti-metabolite, an anti- metabolic therapeutic, a mitotic inhibitor, a growth factor inhibitor, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, or amifostine. In some aspects, the anti-cancer agent targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. [0113] In various aspects, the present disclosure provides a pharmaceutical composition comprising the EGFR-binding peptide as described herein and a pharmaceutically acceptable excipient or diluent.

[0114] In various aspects, the present disclosure provides a pharmaceutical composition comprising the peptide complex as described herein or the peptide-active agent complex as described herein and a pharmaceutically acceptable excipient or diluent.

[0115] In various aspects, the present disclosure provides a method of inhibiting EGFR in a subject, the method comprising administering to the subject a composition comprising the EGFR-binding peptide as described herein and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR.

[0116] In some aspects, the EGFR-binding peptide inhibits activation of the EGFR by disrupting multimerization, dimerization, or heterodimerization of the EGFR on the cell of the subject that expresses EGFR.

[0117] In various aspects, the present disclosure provides a method of selectively depleting a target molecule, the method comprising: a) contacting the peptide complex as described herein to a cell expressing a cellular receptor; b) binding the target-binding peptide to the target molecule under extracellular conditions; c) binding the cellular receptor-binding peptide to the cellular receptor under extracellular conditions; and d) endocytosing the peptide complex, the target molecule, and the cellular receptor into an endocytic or lysosomal compartment, thereby depleting the target molecule.

[0118] In some aspects, the method further comprises: e) dissociating the target-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both under endosomal or lysosomal conditions. In some aspects, the method further comprises: f) degrading the target molecule, thereby further depleting the target molecule. In some aspects, the method further comprises recycling the peptide complex and the cellular receptor to the cell surface.

[0119] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the EGFR-binding peptide as described herein or the pharmaceutical composition as described herein and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition.

[0120] In some aspects, the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, or a panitumumab- resistant EGFR. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0121] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising: a) administering to the subject the EGFR-binding peptide as described herein, the peptide complex as described herein, the peptide-active agent complex as described herein, or the pharmaceutical composition as described herein; b) binding the target-binding peptide under extracellular conditions to a target molecule associated with the disease or condition on a cell of the subject expressing the target molecule and a cellular receptor; c) binding the cellular receptor-binding peptide under extracellular conditions to the cellular receptor on the cell of the subject; and d) endocytosing the peptide complex, the target molecule, and the cellular receptor.

[0122] In some aspects, the method further comprises: e) dissociating the target-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both under endosomal conditions. In some aspects, the method further comprises: f) degrading the target molecule. In some aspects, the target molecule comprises an EGFR. In some aspects, the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, or EGFR mutant T790M. In some aspects, the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

[0123] In some aspects, the disease or condition is a cancer. In some aspects, the cancer expresses EGFR, overexpresses EGFR, or contains mutant EGFR. In some aspects, the cancer is breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, blood-cell-derived cancer, lung cancer, sarcoma, stomach cancer, a gastrointestinal cancer, glioblastoma, head and neck cancer, squamous head and neck cancer, non-small-cell lung cancer, squamous non-small cell lung cancer, pancreatic cancer, ovarian cancer, endometrial cancer, blood cancer, skin cancer, liver cancer, kidney cancer, or colorectal cancer. In some aspects, the cancer is TKI-resistant, cetuximab-resistant, necitumumab-resistant, or panitumumab-resistant. In some aspects, the cancer has one or more of the following: overexpresses EGFR, KRAS mutation, KRAS G12S mutation, KRAS G12C mutation, PTEN loss, EGFR exonl9 deletion, EGFR L858R mutation, EGFR T790M mutation, a cetuximabresistant EGFR, a panitumumab-resistant EGFR, PIK3CA mutation, TP53 R273H mutation, PIK3CA amplification, PIK3CA G118D, TP53 R273H, EGFR C797X mutation, EGFR G724S mutation, EGFR L718Q mutation, EGFR S768I mutation, an EGFR mutation, or a combination thereof. In some aspects, the cancer expresses or has upregulated c-MET, Her2, Her3 that heterodimerizes with EGFR. In some aspects, the cancer is a primary cancer, an advanced cancer, a metastatic cancer, a metastatic cancer in the central nervous system, a primary cancer in the central nervous system, metastatic colorectal cancer, metastatic head and neck cancer, metastatic non-small-cell lung cancer, metastatic breast cancer, metastatic skin cancer, a refractory cancer, a KRAS wild type cancer, a KRAS mutant cancer, or an exon20 mutant non- small-cell lung cancer.

[0124] In some aspects, the method further comprises administering an additional therapy to the subject. In some aspects, the additional therapy is adjuvant, first-line, or combination therapy. In some aspects, the additional therapy targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. In some aspects, targeting of other oncogenic signaling pathways comprises administration of inhibitors of MEK/ERK pathway signaling, PI3K/AKT pathway signaling, JAK/STAT pathway signaling, or WNT/p-catenin pathway signaling. In some aspects, targeting of immune response pathways comprises PD-1/PD-L1 checkpoint inhibition. In some aspects, directly driving an immune response to cancer cells comprises bispecific T cell engagers or chimeric antigen receptor expressing T cells. In some aspects, the targeting disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells comprises administering senolytic agents to a subject. In some aspects, the additional therapy comprises administering fluorouracil, FOLFIRI, irinotecan, FOLFOX, gemcitabine, or cisplatin, irinotecan, oxiplatin, fluoropyrimidine to the subject.

[0125] In some aspects, the method further comprises forming a ternary complex between the selective depletion complex, the target molecule, and the cellular receptor. In some aspects, formation of the ternary complex increases, facilitates, or stabilizes recycling or turnover of the cellular receptor, the target molecule, or both. In some aspects, formation of the ternary complex increases, facilitates, or stabilizes binding of the target molecule to the cellular receptor. In some aspects, the peptide complex binds at higher levels to cells that overexpress the target molecule and the cellular receptor than to cells that have lower levels of the target molecule or the cellular receptor or both.

[0126] In some aspects, the peptide complex has a larger, longer, or wider therapeutic window as compared to an alternative therapy. In some aspects, the alternative therapy is not recycled to the cell surface. In some aspects, the alternative therapy is a lysosomal targeting therapy, a ubiquitin-proteosome system (UPS) targeting therapy, a non-selective therapeutic agent, an existing biologic, or a lysosomal delivery molecule. In some aspects, the peptide complex or the EGFR-binding peptide is administered at lower molar dosage than alternative therapies. In some aspects, the peptide complex or the EGFR-binding peptide binds at higher levels to cancer cells than to normal cells. In some aspects, the peptide complex or the EGFR-binding peptide has a higher antiproliferative effect, a higher target molecule depletion effect, or a higher viability effect on cancer cells than on normal cells in vitro or in vivo. In some aspects, the peptide complex or the EGFR-binding peptide has a larger, longer, or wider therapeutic window than an anti-EGFR antibody or a TKI. In some aspects, the peptide complex or the EGFR-binding peptide has lower toxicity on skin or on keratinocytes than an anti-EGFR antibody or a TKI.

[0127] In various aspects, the present disclosure provides a method of administering a peptide complex or an EGFR-binding peptide to a subject, the method comprising administering the EGFR-binding peptide as described herein, the peptide complex as described herein, the peptide-active agent complex as described herein, or the pharmaceutical composition as described herein.

[0128] In various aspects, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the EGFR-binding peptide as described herein, the peptide complex as described herein, the peptide-active agent complex as described herein, or the pharmaceutical composition as described herein, thereby treating the disease or condition.

INCORPORATION BY REFERENCE

[0129] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0131] FIG. 1 is an image illustrating domain binding in an EGF:EGFR complex. This crystal structure, publicly available and deposited into the RCSB as PDB ID: 1IVO, represents the ectodomains of an active EGFR homodimer (tetrameric due to the presence of two EGF molecules). EGF binding to both EGFR Domain I and Domain III brings Domain II away from Domain IV and into a conformation capable of association with another Domain II. In such a conformation, EGFR is dimerized and activated, leading to phosphorylation and activation of various pathways such as MEK/ERK and PI3K/AKT which leads to intracellular signaling and promote growth of cells, including promoting oncogenic potential and growth of cancer cells. Although the EGF variants herein bind EGFR, they also disrupt the dimerization of EGFR, and hence disrupt, or reduce, or ablate the promotion of growth and hence disrupt, or reduce, or ablate the oncogenic potential of the EGFR on cells. Such EGF variants herein can be used to kill target cancer cells through such disruption or via other means as described herein.

[0132] FIG. 2 illustrates a bar chart showing flow cytometry data for biotinylated soluble EGFR ectodomain and soluble full-length EGFR bound to the surface of a cell expressing wild type EGF (SEQ ID NO: 317), using fluorescent (647 nm excitation) streptavidin as a co-stain. The chart shows that the wild type soluble EGFRvIII ectodomain (i.e., isolated EGFR Domain III, hereafter referred to simply as “EGFRvIII” when describing flow cytometry experiments) does not significantly interact with cell-surface-displayed wild type human EGF, while soluble full- length EGFR ectodomain (hereafter referred to simply as "full-length EGFR” when describing flow cytometry experiments) shows binding. This experiment shows the wild type EGFRvIII ectodomain does not bind to the EGF.

[0133] FIG. 3 illustrates a stepwise flow chart for engineering showing the high-level modification of EGF into a cell growth disrupting variant that binds EGFR but does not signal on the receptor that would also be ready for SDC incorporation or use as a direct EGFR inhibitor. This methodology is a general roadmap to select properties on variant or modified EGF that would be useful alone or in the context of an SDC without describing specific variants themselves. Specific EGF variants are described herein.

[0134] FIG. 4 illustrates the EGF:EGFR interface contacting residues subject to Rosetta design (Domain III) or intentional mutational disruption (Domain I). The publicly available crystal structure of EGFR, deposited into the RCSB as PDB ID: 1IVO, was used to analyze interactions between EGF and EGFR, which reveals EGF residues closely contacting either Domain I (grey font) or Domain III (black font) which are suitable for mutating in order to improve EGF:EGFR Domain III binding strength and/or disrupt EGF:EGFR Domain I binding strength. Specific residues used on the to improve EGF:EGFR Domain III binding strength and/or disrupt EGF:EGFR Domain I binding are depicted using single letter amino acid code. This methodology is a general roadmap to select properties on variant or modified EGF that would be useful alone or in the context of an SDC without describing specific variants themselves. Specific EGF variants are described herein.

[0135] FIG. 5 illustrates cells expressing pooled EGF variants screened for GFP fluorescence-x axis (EGF variant expression) and APC channel fluorescence-y-axis (biotinylated target protein + fluorescent streptavidin binding) using flow cytometry. The inset box in each graph shows that the EGF library has a high proportion of full-length EGFR binding variants (~23% of GFP+ cells), with a much smaller proportion capable of showing appreciable binding to Domain III alone (EGFRvIII, <2% of GFP+ cells), whereas control protein does not show appreciable APC channel fluorescence.

[0136] FIG. 6 illustrates successive enrichment or increase in cells expressing EGF variants capable of binding EGFRvIII using flow cytometry. Cells expressing EGF variants were stained with biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation) at 100 nM each for 15 minutes on ice. GFP fluorescence (EGF variant expression) and APC channel fluorescence (target protein + streptavidin binding) were used to identify GFP+ cells with APC channel fluorescence above background levels, which were flow sorted and collected for further enrichment. The middle and bottom panels show same staining and flow sorting for a further two rounds of flow sorting enrichment.

[0137] FIG. 7 illustrates showing surface displayed EGF variant expression (x axis) and biotinylated EGFRvIII + fluorescent streptavidin binding (y-axis) in cells expressing a single EGF variant, including SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 319, SEQ ID NO: 387, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 369, SEQ ID NO: 372, and SEQ ID NO: 383 measured using flow cytometry.

[0138] FIG. 8 illustrates amino acid sequences of several isolated EGF variants with demonstrable EGFRvIII binding and Domain 1 contact residues (depicted by “X”) identifying mutations (shaded) to Domain I-adjacent residues that do not eliminate binding to Domain III. Four such mutations are the EGF variants depicted as M21R (present in SEQ ID NO: 378), A30W (present in SEQ ID NO: 371), I38D (present in SEQ ID NO: 376), and W49R (present in SEQ ID NO: 383, SEQ ID NO: 384, and SEQ ID NO: 385). Three of these mutations (M21R, 138D, and W49R) replace hydrophobic residues with polar charged residues, potentially disrupting hydrophobic interactions strengthening the EGF:[EGFR Domain I] interaction. The other (A30W) replaces a small side chain with a large, bulky side chain, potentially disrupting EGF:[EGFR Domain I] interaction by steric hindrance.

[0139] FIG. 9 illustrates the EGF:[EGFR Domain I] interface from public crystallography data (deposited in the RCSB as PDB ID: 1IVO), with a focus on four proposed mutations M21R (present in SEQ ID NO: 378), A30W (present in SEQ ID NO: 371), I38D (present in SEQ ID NO: 376), and W49R (present in SEQ ID NO: 383, SEQ ID NO: 384, and SEQ ID NO: 385), and demonstrates their disruption of Domain I binding at specific residues in EGFR Domain I. In each panel, the original EGF residue is in black labeled with black font, while the modeled mutation is in grey labeled in grey font, depicting disrupting hydrophobic interactions strengthening the interaction with Domain 1 or disrupting Domain 1 though steric hindrance.

[0140] FIG. 10 illustrates flow cytometry data from surface-displayed EGF variants that exhibit EGFRvIII binding despite incorporation of one or more Domain I disrupting mutations (x-axis) and their EGFRvIII-binding capability (y-axis). FIG. 10 demonstrates maintenance of EGFR Domain III binding despite incorporation of one or more Domain I disrupting mutations. Variants of the lead EGFRvIII-binding EGF variant (SEQ ID NO: 319) were transfected individually into 293F cells and stained with 50 nM each biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation). A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. EGFRvIII binding was observed for all five variants, with the highest staining seen by EGF84v9 (SEQ ID NO: 458).

[0141] FIG. 11 illustrates a comparison of the wild type EGF and an EGF variant binding characteristics on full length EGFR and EGFRvIII (Domain III only). The binding characteristics of human EGF (SEQ ID NO: 317) control and EGF84v9 (SEQ ID NO: 458). Human EGF (SEQ ID NO: 317) and EGF84v9 (SEQ ID NO: 458) were transfected individually into wells of 293F cells and stained with either 50 nM biotinylated full-length EGFR + fluorescent streptavidin, or 50 nM EGFRvIII + fluorescent streptavidin. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. The variant EGF (SEQ ID NO: 458) could still bind to the full length EGFR in spite of its Domain 1 disruption but demonstrated much stronger binding to EGFRvIII (isolated EGFR Domain III), suggesting Domain I disruption was successful.

[0142] FIG. 12 illustrates, in tabular form, enrichment scores of mutations in a library of EGF84v9 (SEQ ID NO: 458) variants that have high EGFRvIII staining (high DAPI stain) but that lose this binding upon pH 5.5 incubation (low APC stain). Variant abundances were normalized to that of both EGF84v9 (SEQ ID NO: 458) itself and to the variant’s abundance in the initial pre-sort library, enabling evaluation of both overall enrichment/depletion and enrichment/depletion compared to the parent sequence. This double-normalized abundance is log2 -transformed and defined as the variant’s enrichment score; a negative enrichment score represents depletion from the population, while a positive enrichment score represents enrichment, the latter being a byproduct of improved EGFRvIII binding and a high degree of pH 5.5 release. The table shows these enrichment scores, rounded to the nearest integer. Figure discloses SEQ ID NO: 713.

[0143] FIG. 13 illustrates affinity maturation using EGFRvIII binding of EGF84v9 singleton variants analyzed by flow cytometry. While all of the matured variants demonstrated improved EGFRvIII staining compared to SEQ ID NO: 458, four EGF variants were selected for further analysis due to high EGFRvIII staining, including SEQ ID NO: 477, SEQ ID NO: 481, SEQ ID NO: 493, and SEQ ID NO: 494. [0144] FIG. 14 illustrates EGFRvIII binding by EGF84v9 singleton variants SEQ ID NO: 477, SEQ ID NO: 481, SEQ ID NO: 493, and SEQ ID NO: 494 and the parent EGF84v9 (SEQ ID NO: 458) after a 10 minute buffer incubation (“rinse”) at pH 7.4 or pH 5.5. All variants demonstrated substantial reduction of EGFRvIII staining upon pH 5.5 rinse compared to pH 7.4 rinse, but SEQ ID NO: 494 had highest staining retention after pH 7.4 rinse while still very low staining after pH 5.5 rinse. Low or reduced staining at pH 5.5 rinse indicates that the binding strength of the tested singleton EGF variants to EGFRvIII is pH sensitive and the EGFRvIII is dissociated at the lower pH in the context of a bivalent interaction.

[0145] FIG. 15 illustrates EGF variant-based EGFR selective depletion complex (SDC) candidates. All seven molecules contain an Fc domain (homodimeric or heterodimeric knob- and-hole) and one or two domains constituting EGF variant SEQ ID NO: 494. Six also contain one or two domains constituting TfR-binding CDPs of SEQ ID NO: 96, making EGFR SDC candidate molecules (SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 503 paired with SEQ ID NO: 504, SEQ ID NO: 503 paired with SEQ ID NO: 505, and SEQ ID NO: 504 paired with SEQ ID NO: 506). The differences between these SDC candidate molecules are the linker between the TfR-binding domain and the EGF variant domain, and also the variations in valence of EGFR or TfR binding capabilities. A control SDC without TfR-binding capabilities was also designed (SEQ ID NO: 498). Though the control SDC of SEQ ID NO: 498 does not have TfR binding capabilities and should therefore not drive TfR-cycling-dependent EGFR uptake, it does have EGFR binding capabilities and is predicted to bind EGFR without facilitating growth-signal-driving dimerization, which could render SEQ ID NO: 498 into an effective EGFR inhibitor. Ovals marked “N” and “C” represent homodimerization domains of SEQ ID NO: 535; ovals containing a convex or concave semicircular notch and marked “N” and “C” represent heterodimerization domains of SEQ ID NO: 536 and SEQ ID NO: 537, respectively; circles represent TfR-binding variants of SEQ ID NO: 96; 7-pointed stars represent EGF variants of SEQ ID NO: 494; curved and wavey lines represent linkers of SEQ ID NO: 223, SEQ ID NO: 540, SEQ ID NO: 541, or SEQ ID NO: 538.

[0146] FIG. 16 illustrates EGF variant-based EGFR selective depletion complex (SDC) candidates that use PD-L1 as the cellular receptor. All six molecules contain an Fc domain (homodimeric or heterodimeric knob-and-hole), one or two domains constituting EGF variant SEQ ID NO: 494, and one or two domains constituting PD-Ll-binding CDPs of SEQ ID NO: 187, making EGFR SDC candidate molecules (SEQ ID NO: 511, SEQ ID NO: 515, SEQ ID NO: 513, SEQ ID NO: 515 paired with SEQ ID NO: 516, SEQ ID NO: 515 paired with SEQ ID NO: 505, and SEQ ID NO: 506 paired with SEQ ID NO: 516). The differences between these SDC candidate molecules are the linker between the PD-L1 -binding domain and the EGF variant domain, and also the variations in valence of EGFR or PD-L1 binding capabilities. Ovals marked “N” and “C” represent homodimerization domains of SEQ ID NO: 535; ovals containing a convex or concave semicircular notch and marked “N” and “C” represent heterodimerization domains of SEQ ID NO: 536 and SEQ ID NO: 537, respectively; circles represent PD-Ll-binding variants of SEQ ID NO: 187; 7-ponited stars represent EGF variants of SEQ ID NO: 494; curved and wavey lines represent linkers of SEQ ID NO: 223, SEQ ID NO: 540, SEQ ID NO: 541, or SEQ ID NO: 538.

[0147] FIG. 17A-B illustrates the extent and duration of elimination of surface EGFR in the cells by selective depletion complexes (SDC) under different SDC concentrations and treatment regimens.

[0148] FIG. 17A illustrates the extent of elimination of surface EGFR in cells by a selective depletion complex (SDC) of SEQ ID NO: 495 evaluated by analyzing samples by flow cytometry to measure the surface EGFR under different SDC concentrations. A549 cancer cells were grown until approximately 30% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were then left untreated (“Untreated”) or dosed with the SDC comprising SEQ ID NO: 495 at 2 nM, 10 nM, 50 nM, or 200 nM and incubated for a further 24 hours (untreated or in the presence of the SDC). After 24 hours, cells were rinsed, collected, stained with an antibody vs EGFR and a fluorescent co-stain, and subjected to flow cytometry to quantitate fluorescent signal corresponding to the amount of surface EGFR per cell in the population. The data for each population were averaged and plotted relative to the untreated population (Surface EGFR (% of untreated ± 95% confidence interval (CI)).

[0149] FIG. 17B illustrates the extent and duration of elimination of surface EGFR in the cells by a selective depletion complex (SDC) evaluated by analyzing samples by flow cytometry to measure the surface EGFR under different SDC treatment regimens. A549 cancer cells were grown until approximately 20% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were then left untreated or dosed with 10 nM of the SDC comprising SEQ ID NO: 495 and incubated for a further 24 hours. After 24 hours, the media in the wells was replaced with fresh media. Three treatments were then performed and analyzed over the next 24 hours (summarized in the table in FIG. 17B): (1) the cells that had been subjected to treatment with the SDC comprising SEQ ID NO: 495 for 24 hours were then left untreated for an additional 24 hours (referred to as the “Withdrawal” sample, as it was subjected to 24 hour treatment with the SDC followed by a 24 hour in untreated media (that is, treatment withdrawal of the SDC)), (2) previously-untreated cells were dosed with 10 nM of the SDC comprising SEQ ID NO: 495 for 24 hours (referred to as the “24 hr” sample, as its surface EGFR was analyzed immediately after a 24 hour SDC treatment, and (3) a third sample (“Untreated”) that was left untreated during the previous 24 hours was still left untreated for an additional 24 hours. After this second 24 hour incubation, cells in all samples were rinsed, collected, stained with an antibody vs EGFR and a fluorescent co-stain, and subjected to flow cytometry to quantitate fluorescent signal corresponding to the amount of surface EGFR per cell in the population. The data for each population were averaged and plotted relative to the untreated population (Surface EGFR (% of untreated ± 95% confidence interval (CI)).

[0150] FIG. 18 illustrates the uptake of soluble EGFR into the cells evaluated by analyzing samples by flow cytometry to measure the soluble EGFR uptake under different SDC treatment regimens. Hl 975 cancer cells were grown until approximately 40% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. After this point, cells underwent one of three treatments to assess uptake of soluble biotinylated EGFRvIII (sEGFR; SEQ ID NO: 527). Each of the three treatments are summarized in the table in FIG. 18. Treatment (1), referred to as “No SDC”, incubated the cells for two hours in media with 20 nM sEGFR and 20 nM unlabeled monovalent streptavidin. After this 2 hour treatment, cells are rinsed twice with PBS and then incubated for 24 hours in media with 10 nM sEGFR and 10 nM fluorescent streptavidin. Treatment (2), referred to as “SEQ ID NO: 495 Pre-treat”, incubated the cells for 2 hours in media with 20 nM sEGFR, 20 nM unlabeled monovalent strepatavidin, and 5 nM of the SDC comprising SEQ ID NO: 495. This treatment first exposed cells to sEGFR-saturated SDC molecules. After this 2 hour treatment, cells were rinsed twice with PBS and then incubated for 24 hours with 10 nM sEGFR and 10 nM fluorescent streptavidin to assess entry of the fluorescent sEGFR into the cells. Treatment (3), referred to as “SEQ ID NO: 495 24 hr”, was the same treatment as “SEQ ID NO: 495 Pre-treat”, except the second incubation (24 hours with 10 nM sEGFR and 10 nM fluorescent streptavidin) also included 5 nM SDC comprising SEQ ID NO: 495.

[0151] FIG. 19 illustrates the assessment of disruption of EGFR signaling by an EGF -variant selective depletion complex (SDC) as well as the absence of EGFR activation by molecules that incorporate the EGF variant (designed to bind EGFR but not activate it) analyzed by Western blot. A549 cancer cells were grown until approximately 30% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were left untreated or dosed with either the SDC comprising SEQ ID NO: 495 or the control comprising SEQ ID NO: 498 at 10 nM and incubated for a further 24 hours. After 24 hours, and without exchanging media, cells were either left as-is or exposed to 50 ng/mL EGF (SEQ ID NO: 317). Cells were then lysed in buffer containing protease/phosphatase inhibitors. Lysate protein content was quantitated by bicinchoninic acid (BCA) assay and was ran on an SDS-PAGE gel prior to electrophoretic transfer to a polyvinylidine difluoride (PVDF) membrane and subsequent blocking and blotting with antibodies against actin, total EGFR, or phospho-Y1068 (pY1068) EGFR, the latter being a marker for ligand-activated EGFR. A fluorescent secondary antibody provides signal that can be read on a scanner.

DETAILED DESCRIPTION

[0152] Described herein are compositions and methods for selective depletion of an EGFR target molecule using cellular endocytic pathways (e.g., transferrin receptor-mediated endocytosis). Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in cell signaling. EGFR signaling regulates cell growth and survival, and upregulation of EGFR is implicated in many types of cancer. EGFR proteins are regularly cycled through production, use, and degradation, and their degradation is typically within the endosomal-lysosomal pathway. In this pathway, endocytic vesicles containing material taken up from extracellular space as well as embedded membrane proteins become acidified and fuse with or enter lysosomes containing enzymes that degrade such proteins. Selective removal of certain cell proteins such as EGFR proteins, either from circulation or disease-associated tissues, such as by removing the proteins from the cell surface or from soluble forms, optionally with selective delivery to the lysosome, can be used to treat disease conditions, including diseases resulting from over-expression or mutations in EGFR. Alternatively or in addition, the peptide complexes described herein for selective depletion of a target molecule, also referred to as “selective depletion complexes”, can be used to deliver an administered therapeutic drug to a cell or an endosomal or lysosomal compartment in cells or tissues with increased EGFR expression, for example to treat lysosomal storage diseases like Gaucher’s Disease (deficiency of glucocerebrosidase) or Pompe Disease (deficiency of a-glucosidase) or a disease associated with EGFR (e.g., a cancer). A therapeutic molecule (e.g., a lysosomal enzyme for an enzyme replacement therapy or a chemotherapeutic agent) can be administered with a selective depletion complex comprising a target-binding peptide that binds the therapeutic molecule, thereby delivering the therapeutic molecule to the endosome or lysosome. In some embodiments, a selective depletion construct can function as a selective delivery complex and facilitate delivery of active enzymes to an endosome or lysosome. For example, a lysosomal enzyme can be delivered using a selective depletion complex and can retain enzymatic activity in the endosome or lysosome. Administration of a lysosomal enzyme in combination with a selective depletion complex comprising a targetbinding peptide that binds the lysosomal enzyme can increase the therapeutic response per dose of enzyme administered relative to administration of the lysosomal enzyme alone. For either selective depletion of target proteins from a cell, or delivery of lysosomal proteins, lysosomal delivery could be accomplished by taking advantage of existing protein uptake and recycling mechanisms, and engineering of pH-dependent binding domains into target-binding molecules. [0153] A unique example of an endocytic pathway that can be used for selective depletion of target molecules is via transferrin receptor (TfR) internalization and trafficking, which is normally used for transferrin recycling via transferrin receptor (TfR) for iron delivery to cells and tissues. Transferrin is known as a serum chaperone for iron ions destined for redox sensitive intracellular enzymes. Iron-loaded transferrin (holo-transferrin) delivers iron to cells via specific binding to TfR, which is then trafficked to endosomes, where the pH is reduced by native proton pumps. Under acidic conditions, transferrin loses its iron binding affinity, releasing iron inside the cell, but maintains its TfR-binding affinity. The TfR:transferrin complex is natively recycled back to the cell surface, exposing transferrin to neutral pH conditions. Transferrin unbound by iron (apo-transferrin) no longer has TfR affinity under neutral pH conditions at the cell surface and is released back into circulation to pick up more iron, and repeat the process, in what is essentially a catalytic process for iron delivery to cells.

[0154] The compositions and methods of this disclosure exploit the transferrin receptor endocytic and recycling pathways to selectively deplete target molecules (e.g., EGFR) from the cell surface, disrupt EGFR multimerization, and/or to selectively deplete and deliver target molecules (e.g., EGFR) to endocytic vesicles for lysosomal degradation. The compositions and methods of this disclosure can be used to selectively deplete or degrade EGFR target receptor proteins or over-expressed in disease via this pathway. As a result of depleting target molecules from the cell surface, and/or through lysosomal degradation of the target receptors (e.g., EGFR), the compositions and methods described effectively reduce, diminish, eliminate, or deplete the target receptors from the cell surface which has many applications in medicine as described herein. Selective depletion complexes of the present disclosure comprising a TfR-binding peptide (e.g., a TfR-binding cystine-dense peptide) coupled to a target-binding peptide (e.g., a target-binding EGF variant) can recruit a target molecule to the TfR by binding to both the TfR (via the TfR-binding peptide) and to the target molecule (via the target-binding peptide). Upon endocytosis, the TfR can carry the selective depletion complex and the target molecule into the endocytic vesicle. In some embodiments, the TfR-binding peptide of the selective depletion complex can have high affinity for TfR at extracellular pH (about pH 7.4) to maturing endosomal pH (about pH 5.5), inclusive. The TfR-binding peptide can maintain its affinity for TfR upon internalization and as the endosomal compartment acidifies. The target-binding peptide of the selective depletion complex can have higher affinity for the target molecule at extracellular pH and lower affinity for the target molecule at a lower endosomal pH. Inside the endocytic vesicle, the selective depletion complex can remain bound to TfR and release the target molecule upon acidification of the endosome. Once the target molecule is released, the selective depletion complex can remain bound to TfR while TfR is recycled to the cell surface to be reloaded with another target molecule, and the target molecule can remain in the endosome and in some embodiments the target molecule is further delivered to a lysosome and degraded. In some embodiments, the TfR-binding peptide of the selective depletion complex can have higher affinity for TfR at extracellular pH and lower affinity for the target molecule at a lower endosomal pH. Inside the endocytic vesicle, the selective depletion complex can release from TfR upon acidification of the endosome. In some embodiments, the dissociation rate of the selective depletion complex from the target while in the endosome is faster than the rate of endosome recycling back to the cell surface. In this case, the target may be released from the selective depletion complex regardless of any or no variation in affinity to the target as a function of pH.

[0155] The methods of the present disclosure can comprise contacting a cell (e.g., a cell expressing TfR) with a selective depletion complex (e.g., a molecule comprising a TfR-binding peptide and a target-binding peptide). The selective depletion complex can recruit target molecules into endocytic vesicles via transferrin receptor-mediated (TfR-mediated) endocytosis. The target molecule can be released in the endocytic vesicle, and it may be further delivered to the lysosome and degraded. The selective depletion complex can remain bound to the TfR and can remain bound to TfR as TfR is recycled to the cell surface. Such methods can be used to deplete a target molecule (e.g., EGFR), such as a molecule associated with a disease or a condition (e.g., associated with cancer). For example, the methods of the present disclosure can be used to selectively deplete EGFR that is over-expressed, contains a disease-associated mutation (e.g., a mutation causing constitutive activity, resistance to treatment, or dominant negative activity), or accumulates in a disease or a condition. It is understood that selective depletion of a target molecule includes the depletion of the selected target from the cell surface or soluble target in circulation, each of which could result in a therapeutic effect of the selective depletion complex.

[0156] In some embodiments, the presently described selective depletion complex can comprise peptide conjugates, peptide complexes, peptide constructs, fusion peptides, or fusion molecules such as linked by chemical conjugation of any molecule type, such as small molecules, peptides, or proteins, or by recombinant fusions of peptides or proteins, respectively (e.g., a peptide construct or a peptide complex). The terms “fusion peptide” and “peptide fusion” are used interchangeably herein. In some embodiments, the peptide constructs or peptide complexes can be produced biologically or synthetically. Thus, in some cases, a selective depletion complex can comprise a TfR-binding peptide domain linked to another molecule or group of molecules such as small molecules, peptides, or proteins or other macromolecules such as nanoparticles. [0157] In some embodiments, the presently described selective depletion complexes can be peptide complexes comprising one or more TfR-binding peptides as described herein conjugated to, linked to, or fused to. or complexed with one or more target-binding peptides (e.g., one or more EGFR-binding peptides), one or more active agents (e.g., therapeutic agents, detectable agents, or combinations thereof), or combinations thereof. Selective depletion complexes as described herein can include chemical conjugates and recombinant fusion molecules. In some cases, a chemical conjugate can comprise a TfR-binding peptide as described herein that is chemically conjugated to or linked to another peptide (e.g., an EGFR target-binding peptide), a molecule, an agent, or a combination thereof. Molecules can include small molecules, peptides, polypeptides, proteins, or other macromolecules (e.g., nanoparticles) and polymers (e.g., nucleic acids, polylysine, or polyethylene glycol). In some cases, a TfR-binding peptide of the present disclosure is conjugated to another peptide or a molecule via a linker. Linker moieties can include cleavable (e.g., pH sensitive or enzyme-labile linkers) or stable linkers. In some embodiments, a peptide complex is a fusion molecule (e.g., a fusion peptide or fusion protein) that can be recombinantly expressed, and wherein the fusion molecule can comprise one or more TfR-binding peptides fused to one or more other molecules peptides, polypeptides, proteins, or other macromolecules that can be recombinantly expressed.

[0158] The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can have a therapeutic effect at a lower dose or a longer lasting therapeutic effect as compared to lysosomal delivery molecules that are degraded and not recycled to the cell surface. Rather than being degraded in the lysosome, the selective depletion complexes of this disclosure can be recycled back to the cell surface to “reload” with the target molecule, meaning that the potential for one selective depletion complex of this disclosure can drive the degradation of multiple target molecules with a potentially catalytic effect. The selective depletion complex may also continue to have depletion activity even when the selective depletion complex is no longer present in serum but is present on or in a cell. A lysosomal delivery molecule that is not recycled to the cell surface can itself be degraded or can accumulate in the lysosome without being re-used or “reloaded”. The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can have a larger (e.g., longer or wider) therapeutic window (i.e., the dosage above which a therapeutic pharmacodynamic response is observed but below which toxicity is observed) or a higher potency or a longer duration of effect as compared to lysosomal delivery molecules that are not recycled to the cell surface. The therapeutic window of a drug (e.g., a selective depletion complex of the present disclosure) is the dose range at which the drug is effective without having unacceptable toxic effects. The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can be used with less risk of toxicity. The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can be used (e.g., administered) at lower molar dosage than alternative therapies (e.g., lysosomal delivery molecules) that are not recycled to the cell surface. Because of the selectivity and re-usable nature of the selective depletion complexes of this disclosure in the cell, as therapeutic agents they are advantageously not depleted as rapidly as non-recyclable delivery compositions targeted to lysosomes which are depleted as they are used. The selective depletion complexes can be more concentrated on tissues that express the receptor, such as TfR, or the target (such as EGFR) at higher levels than other tissues; as such, the selective depletion complexes and their effects can be more concentrated on diseased tissues that overexpress the applicable receptor or the target or both compared to normal or healthy tissues. Many solid tumors express TfR at high levels and thus selective depletion complexes that bind TfR may be more concentrated on solid tumor tissues, concentrating their depletion effect on the tumor tissues. Many human tumors express EGFR at high levels (e.g., the lung, head and neck, colon, pancreas, breast, ovary, bladder and kidney, and in glioma) and thus selective depletion complexes that bind EGFR may be more concentrated on such tumor tissues, concentrating their depletion effect on the tumor tissues. Moreover, because of the selectivity and recycling aspect of the selective depletion complexes of this disclosure (e.g., selective depletion complexes comprising a receptor-binding peptide and an EGFR target-binding peptide), as therapeutic agents they are advantageously less toxic than non-selective therapeutic agents. This is particularly advantageous for applications in cancer, where therapeutic agents can be non-selective and highly toxic and exhibit detrimental side effects on normal cells, organs, and tissues, or require lower than effective therapeutic doses less able to reduce, cure, ablate disease.

[0159] In other embodiments, a selective depletion complex can bind the PD-L1 receptor, rather than TfR, to enable uptake of the target and recycling of the selective depletion complex. PD-L1 is expressed by cells such as solid tumor cells, pancreatic beta cells, and certain cells of the immune system. PD-L1 can be taken up by endosomes, and then recycled back up to the cell surface. PD-L1 can co-localize with CMTM6 in recycling endosomes, where CMTM6 prevents PD-L1 from being targeted for lysosomal degradation. A selective depletion complex can bind to PD-L1 and to a target, such as EGFR, that is targeted for depletion. When PD-L1 is endocytosed into the cell, the target can be depleted from the cell surface. The selective depletion complex may recycle back up to the cell surface along with PD-L1 and the target may remain in the endosome and may proceed to the lysosome and be degraded. Selective depletion complexes that use PD-L1 for uptake have the potential to function in all the ways that selective depletion complexes that use TfR for uptake may function. Use of PD-L1 as a selective depletion complex receptor may permit selective targeting of selective depletion complexes to solid tumor cells, as PD-L1 is expressed in high levels on some solid tumors but is otherwise not commonly expressed in adult tissues except for some specific cell populations (e.g., certain cells of the immune system or pancreatic beta cells).

[0160] The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can have less immunogenicity than an alternative therapy (e.g., a lysosomal delivery molecule) that contains sugars, glycans, polymers containing sugar-like molecules, or other derivatives. A selective depletion complex of this disclosure can have less immunogenicity than an alternative therapy (e.g., a lysosomal delivery molecule) that targets the mannose-6-phosphate receptor, folate receptor, or the asialoglycoprotein receptor (ASGPR). A selective depletion complex of this disclosure can be manufactured by a single recombinant expression and can have improved manufacturing yield, purity, cost, or manufacturing time than a molecule that has multiple synthetic steps to generate a ligand for mannose-6-phosphate receptor, folate receptor, or the asialoglycoprotein receptor (ASGPR). A selective depletion complex of this disclosure can have a greater therapeutic effect or a lower therapeutic dose due to the ability to design the linker for maximal ability to bind for the receptor (e.g., TfR) and the target molecule (e.g., EGFR) at the same time, including of the target molecule is bound in the cell surface. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure can have fewer epitopes to trigger an adaptive immune response, resulting in reduced immunogenicity as compared to TfR-binding antibody-based therapeutics. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure can exhibit more facile and less disruptive incorporation of active agents into protein fusion complexes as compared to TfR-binding antibody-based therapeutics. The TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure can have a smaller surface area, resulting in lower risk for off-target-binding, as compared to TfR-binding antibody-based therapeutics. The TfR- binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure can be formulated at a higher molar concentration than TfR-binding antibody-based therapeutics due to their lower molecule weight, lower hydrodynamic radius, or lower molar solution viscosity.

[0161] The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can, in some embodiments, cross the blood brain barrier. The selective depletion complexes of this disclosure may be able to transcytose across endothelial cells of the blood-brain barrier and thereby reach the central nervous system (CNS), brain, and associated cells. By this mechanism, the selective depletion complexes of this disclosure may be able to deplete targets that are in the CNS including tumors that are present in the brain. Because the blood-brain barrier excludes the great majority of molecules from the brain, alternative therapies (such as lysosomal delivery methods targeting receptors not known to facilitate blood brain barrier transcytosis or ubiquitin-proteosome system (UPS) targeting therapies) may be unable to reach targets in the CNS.

[0162] The selective depletion complexes of this disclosure (e.g., complexes comprising a receptor-binding peptide and an EGFR target-binding peptide) can function with a wide range of linker lengths. Selective depletion complexes of this disclosure can have a range of short to long linkers between the receptor-binding portion and the target binding portion of the SDC. and the structure of the SDC does not necessarily require close association of the target and the receptor in order cause depletion of the target so long as it forms a ternary complex with the receptor and target that is endocytosed when the receptor is endocytosed. Alternative approaches, such as inducing a ternary complex between a cell surface E3 ligases and a cell surface target to increase or facilitate ubiquitination of the cell surface target and thereby leading to depletion of the target, typically require the target and the cell surface E3 ligase to come in close physical proximity in a suitable orientation in order for the target to be ubiquitinated. Of the dozens of cell surface E3 ligases, most are only expressed on some tissues and may not be expressed at high levels on solid tumors. Many targets (e.g., 55% of cell surface targets) may not have a suitable ubiquitination site accessible by the ligase domain. Because the ubiquitination domain of cell surface E3 ligases is on the intracellular portion of the ligase, it is unable to ubiquitinate extracellular soluble targets and thus the use of E3 ligases does not facilitate ubiquitin-mediated depletion of soluble targets for depletion. The selective depletion complexes of this disclosure do not require such proximity constraints and have been demonstrated to promote the cellular uptake of soluble targets (e.g., soluble EGFRvIII) and hence are an improvement on such prior systems.

[0163] In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure exhibit lower on- target toxicity than an anti-TfR antibody or other therapeutic agents when administered to a subject at the same molar dose or at a similarly effective dose. In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides exhibit lower off-target toxicity than an antibody or other therapeutic agent when administered to a subject at the same molar dose or a similarly effective dose. For example, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR- binding fusion peptides of this disclosure can be administered to a subject at about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold higher molar dose than an antibody while providing similar or lower observed toxicity. In some embodiments, the TfR-binding peptides, TfR-binding peptide conjugates, or TfR-binding fusion peptides of this disclosure exhibit higher efficacy than an anti-TfR antibody or other therapeutic agent when administered to a subject at the same dose by weight as the anti-TfR antibody or other therapeutic agent. The TfR-binding peptides of the present disclosure, when fused to a half-life extending moiety (e.g., Fc, SA21, PEG), can be delivered at even lower doses while preserving activity and efficacy and, thus, is far superior to administering an anti-TfR antibody or other therapeutic agent.

[0164] In some embodiments, the present disclosure provides peptides (e.g., CDPs, knotted peptides, or hitchins), chemical conjugates (e.g., comprising one or more TfR-binding peptides and one or more active agents), or recombinantly expressed fusion molecules (e.g., comprising one or more TfR-binding peptides and one or more active agents) that bind to TfR, PD-L1, other receptors, or the target. The TfR-binding peptides can be cystine-dense peptides (CDPs). The terms “peptides”, “miniproteins”, “proteins”, “CDPs”, “TfR-binding peptides,” “TfR-binding CDPs,” “TfR-binding peptides,” and “engineered TfR-binding peptides” are used interchangeably herein. The binding of peptides described in the present disclosure to TfR can facilitate transcytosis of the selective depletion complex, peptide, peptide complex or peptide construct (e.g., fusion protein, or peptide conjugated to, linked to, complexed with, or fused to an agent) across a cell barrier (e.g., the BBB). The binding of peptides described in the present disclosure to TfR can facilitate endocytosis of the selective depletion complex, peptide, or peptide complex in any cell that expresses TfR, or in cell that express TfR at higher levels, including some cancer cells, hepatic cells, spleen cells, and bone marrow cells. Also disclosed herein is the use of a mammalian surface display screening platform to screen a diverse library of CDPs and identify CDPs that specifically bind to human TfR. Such identified peptides can be modified to improve binding to TfR and used in selective depletion complexes as the peptide or peptide complex that binds TfR and is recycled to the cell surface (e.g., the pH-independent TFR-binding CDP). Also disclosed herein is the use of a mammalian surface display screening platform to screen a diverse library of CDPs and identify CDPs that specifically bind to a target molecule that is desired to be degraded. Such identified peptides can be optimized for binding to a selected target molecule and used in selective depletion complexes as the peptide or peptide complex that binds such selected target molecule and is released in the endosome for degradation within the cell (e.g., the pH-dependent target-binding EGF variant as shown in FIG. 14). Further affinity maturation can be subsequently implemented to produce an allelic series of TfR-binding CDPs or target-binding CDPs as appropriate with varying affinities. In some embodiments, TfR-binding CDPs or target-binding CDPs are identified, and binding can be determined by crystallography or other methods. Peptides of the present disclosure can have cross-reactivity across species. For example, the peptides disclosed herein, in some cases, bind to human and murine TfR. Peptides disclosed herein can accumulate in the CNS and can penetrated the BBB via engagement of the TfR, following intravenous administration. Disclosed herein are TfR-binding CDPs for use as therapeutic delivery agents in oncology, autoimmune disease, acute and chronic neurodegeneration, and pain management. Delivery of active or pharmaceutical agents via TfR-binding CDP can be advantageous over conventional anti-TfR antibodies due to simpler manufacturing (peptides can be made via biologic or synthetic means), improved stability, improved therapeutic window (e.g., a larger, longer, or wider therapeutic window), and smaller size (less potential for steric hindrance of cargo activity). Thus, the methods and compositions of the present disclosure can provide a solution to the problem of effectively transporting cargo molecules (e.g., therapeutic and/or diagnostic small molecules, peptides or proteins) into the CNS (e.g., the brain). For example, the peptides of the present disclosure aid in drug delivery to tumors located in the brain.

[0165] In some embodiments of the present disclosure, a diverse library of CDPs, knotted peptides, hitchins, or peptides derived from knotted peptides or hitchins can be used in combination with a mammalian surface display screening platform is used to identify peptides that specifically bind to human TfR or PD-L1 or other receptors desired for recycling or to a target molecule desired for degradation. (See e.g., Crook et al. (2017) Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets. Nat Commun 8:2244). In some embodiments, a diverse library of CDPs, knotted peptides, hitchins, or peptides derived from knotted peptides or hitchins is mutagenized from endogenous peptide sequences to provide novel peptide sequences. Once TfR-binding or target-binding peptides have been identified, affinity maturation (e.g., site-saturation mutagenesis) can be performed to produce an allelic series of binders with varying (e.g., improved) affinities for TfR or a target molecule. These techniques can be used in combination with various other analytical methods (e.g., crystallography or spectroscopy) in order to determine the nature of peptide-receptor interaction (e.g., critical amino acid residues for receptor binding etc.). In some cases, the peptides of the present disclosure are developed to bind human TfR.

[0166] In some embodiments, the engineered peptides of the present disclosure (e.g., histidine- containing or histidine-enriched target-binding peptides) can have a high target-binding affinity at physiologic extracellular pH (e.g., a pH from about pH 7.2 to about pH 7.5, a pH of from about pH 6.5 to about 7.5, or a pH of from about pH 6.5 to about pH 6.9) but a significantly reduced binding affinity at lower pH levels such as endosomal pH of about 6.5, about 6.0, about 5.8, or about 5.5. Extracellular pH can be, for example pH 7.4. Extracellular pH can also be lower, including in the tumor microenvironment, such as pH 7.2, 7.0, or 6.8. In some embodiments, for example in a tumor environment, extracellular pH can be from about pH 6.5 to about pH 6.9. Upon endocytosis, the endosome undergoes a decrease in pH. Endosomal pH can decrease by the action of proton pumps or by merging with other vesicles with lower pH. The pH can decrease to 7.0, and then to 6.5, and then to 6.0, and then to 5.8, and then to 5.5 or lower. Some endosomes are called early endosomes and can have a pH around 6.5. Some of these endosomes become recycling endosomes. Some endosomes are called late endosomes and can have a pH around 5.5. Some endosomes become or merge with lysosomes, where the pH can be 4.5. Enzymes and other factors in the lysosome can cause degradation of the contents of the lysosome. In some embodiments, the target-binding peptides release in the endosome at about pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. In some embodiments, the target-binding peptide may release at any point during the endosomal maturation process upon a decrease in pH following endocytosis. In some cases, histidine scans and comparative binding experiments can be performed to develop and screen for such peptides. In some embodiments, an amino acid residue in a peptide of the present disclosure is substituted with a different amino acid residue to alter a pH-dependent binding affinity to the target molecule or to TfR or other receptors. The amino acid substitution can increase a binding affinity at low pH, increase a binding affinity at high pH, decrease a binding affinity at low pH, decrease a binding affinity at high pH, or a combination thereof. For example, a peptide that has high affinity to TfR and used in selective depletion complexes as the peptide or peptide complex that binds TfR for recycling to the cell surface can be a pH-independent TfR-binding peptide (e.g., a pH-independent TfR- binding CDP) such that it is not released in the endosome. In some embodiments, the TfR- binding peptide can remain bound to TfR as the ionic strength of the endosomal compartment increases upon acidification of the endosome. In some embodiments the TfR-binding peptides are stable at endosomal pH, and do not release in the endosome for example under acidic conditions, such as pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. Conversely, a peptide that has high affinity for binding to a selected target molecule and used in selective depletion complexes as the peptide or peptide complex that binds such selected target molecule and is released in the endosome for degradation within the cell can be a pH-dependent target-binding CDP such that it is released in the endosome. In some embodiments, a target-binding peptide can release the target molecule as the ionic strength of the endosomal compartment increases upon acidification of the endosome. In some embodiments the target-binding peptides are less stable at endosomal pH, and release wholly or in part in the endosome for example under acidic conditions, such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. In some cases, the TfR-binding peptides of the present disclosure can be optimized for improved intra- vesicular (e.g., intra-endosomal) function while retaining high TfR binding capabilities. In some embodiments, the target-binding peptide may not release at any point during the endosomal maturation process, for example using designs that are not pH-sensitive (i.e., are pH-independent) to release the target molecule in the endosome or lysosome, but the selective depletion complex still results in selective depletion of the target molecule from the cell surface or soluble target molecule in circulation. Exemplary TfR-binding peptides of the present disclosure are shown in TABLE 1 with amino acid sequences comprising SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64.

[0167] Described herein are, in some embodiments, peptides and peptide complexes and methods of screening for peptides and peptide complexes that bind to a protein or molecule of interest, such as TfR, or bind to a target molecule for depletion, or both. Compared to wild type or endogenous molecules such as transferrin, the methods and compositions as described herein can provide peptides with improved TfR-binding capabilities, or peptides that exhibit improved transport capabilities across the BBB, or any combination thereof. In some cases, the presently described peptides efficiently transport cargo molecules (e.g., target-binding molecules) across endothelial cell layers (e.g., the BBB) or epithelial layers. In some embodiments, the TfR- binding peptides of the present disclosure bind to a TfR and promote vesicular transcytosis. In some cases, the TfR-binding peptides of the present disclosure bind to a cell that overexpress a TfR (e.g., a cancer cell) and promotes uptake of the peptide by the cell. In some aspects, a TfR binding peptide or peptide complexes as described herein promotes vesicular transcytosis and uptake by a TfR-overexpressing cell such as a cancer, or a combination thereof. In some cases, the TfR-binding peptides of the present disclosure facilitate TfR-mediated endocytosis of a selective depletion complex and a target molecule.

[0168] The TfR-binding peptides of the present disclosure can bind TfR of different species including human, monkey, mouse, and rat TfR. In some cases, variations or mutations in any of the amino acid residues of a TfR-binding peptide can influence cross-reactivity. In some cases, variations or mutations in any of the amino acid residues of a TfR-binding peptide that interact with the bindings site of TfR can influence cross-reactivity.

[0169] Described herein are peptides, including, but not limited to, designed or engineered peptides, recombinant peptides, and cystine-dense peptides (CDPs)/small disulfide-knotted peptides (e.g., knotted peptides, hitchins, and peptides derived therefrom), that can be large enough to carry a cargo molecule while retaining the ability to bind a target protein with high affinity (e.g., TfR), but yet small enough to access cellular tissues, such as the center of cell agglomerates (e.g., solid tumors). In some cases, the peptides as described herein carry cargo molecules across the BBB into the CNS (e.g., the parenchyma) via vascular transcytosis. In some cases, the transcytosis is TfR-mediated.

[0170] Further described herein are methods and compositions for determining the nature of peptide-receptor interactions (e.g., using X-ray crystallography) as well as their pharmacodynamic and pharmacokinetic properties in vivo, including accumulation in the CNS (e.g., brain), or other affected organs and tissues. Some of the peptides described herein have the ability to target molecule and accumulate in tumor cells. In some cases, the tumor cells overexpress TfR, EGFR, or both. In some aspects, the peptides of the present disclosure have high in vivo stabilities, e.g., high protease stability, high tolerability of reducing agents such as glutathione (GSH), and tolerate elevated temperatures (e.g., up to 95 °C).

[0171] The present disclosure provides, in some embodiments, a peptide or protein design approach based on the 3D protein or receptor structure for identifying peptides or proteins capable of binding such receptor. In some cases, the receptor is a transferrin receptor.

[0172] As used herein, the abbreviations for the natural L-enantiomeric amino acids are conventional and are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai). Typically, Xaa can indicate any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0173] Some embodiments of the disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analogue thereof. When an amino acid sequence is represented as a series of three-letter or one-letter amino acid abbreviations, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard usage and convention.

[0174] The terms “peptide”, “polypeptide”, “miniprotein”, “protein”, “hitchin”, “cystine-dense peptide”, “knotted peptides” or “CDP” can be used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, “peptides”, “polypeptides”, and “proteins” can be chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (e.g., amino terminal, or N-terminal) therefore can have a free amino group, while the terminal amino acid at the other end of the chain (e.g., carboxy terminal, or C-terminal) can have a free carboxyl group. As used herein, the term “amino terminus” (e.g., abbreviated N-terminus) can refer to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (e.g., imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” can refer to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether or thioether as opposed to an amide bond.

[0175] As used herein, the term “peptide construct” can refer to a molecule comprising one or more peptides of the present disclosure that can be conjugated to, linked to (including by complexation), or fused to one or more peptides or cargo molecules. In some cases, cargo molecules are active agents. The term “active agent” can refer to any molecule, e.g., any molecule that is capable of eliciting a biological effect and/or a physical effect (e.g., emission of radiation) which can allow the localization, detection, or visualization of the respective peptide construct. In various embodiments, the term “active agent” refers to a therapeutic and/or diagnostic agent. A peptide construct of the present disclosure can comprise a TfR-b inding peptide that is linked to one or more active agents via one or more linker moieties (e.g., cleavable or stable linker) as described herein.

[0176] As used herein, the term “peptide complex” can refer to one or more peptides of the present disclosure that are fused, linked, conjugated, or otherwise connected to form a complex. In some cases, the one or more peptides can comprise a TfR-b inding peptide, a target-binding peptide, a half-life modifying peptide, a peptide that modifies pharmacodynamics and/or pharmacokinetic properties, or combinations thereof. For example, a peptide complex comprising a TfR-b inding peptide and a target-binding peptide can be referred to herein as a selective depletion complex.

[0177] As used herein, the terms “comprising” and “having” can be used interchangeably. For example, the terms “a peptide comprising an amino acid sequence of SEQ ID NO: 32” and “a peptide having an amino acid sequence of SEQ ID NO: 32” can be used interchangeably.

[0178] As used herein, and unless otherwise stated, the term “TfR” or “transferrin receptor” is a class of protein used herein and can refer to a transferrin receptor from any species (e.g., human or murine TfR or any human or non-human animal TfR). In some cases, and as used herein, the term “TfR” or “transferrin receptor” refers to human TfR (hTfR) and can include TfR or any of the known TfR homologs or orthologs, including TfRl, TfR2, soluble TfR, or any combination or fragment (e.g., ectodomain) thereof.

[0179] As used herein, the terms “endosome,” “endosomal,” “endosomal compartment,” or “endocytic pathway” can be used interchangeably and may refer to any one or more components of the intracellular endosomal network or trans-Golgi network (TGN) that allows for the vesicular transcytosis or trafficking and transfer of peptides and cargoes between distinct membrane-bound compartments within a cell, including lysosomal degradation as well as recycling to the cell surface. It is understood that such pathway involves and includes the maturation and transition of vesicles commonly referred to as transport vesicles or early endosomes to late endosomes to lysosomes, and that endosomal compartment acidity increases upon acidification of the endosome throughout the maturation process. Lysosomes serving as the last vesicle in the matured endocytic pathway typically contain hydrolytic enzymes which digest the contents of the late endosomes. Other endosomes continue to a pathway of recycling endosomes, where the contents are recycled back to the cell surface.

[0180] As used herein “pH-independent,” when used in reference to a molecule or moiety, refer means that as the endosomal compartment is acidified, the binding affinity of the molecule or moiety to its target molecule does not change sufficiently to enable dissociation in the endosome with the target molecule. For example, the referenced molecule or moiety has the same or similar affinity to its target molecule at extracellular pH and at an endosomal pH. It is also understood that pH-independent molecules or moieties do not include pH-dependent molecules or moieties, since the binding affinity of pH-dependent molecules or moieties to its target molecule changes as it enters and proceeds through the endosomal pathway, for example, to enable dissociation in the endosome with the target molecule to some degree, or the referenced molecule or moiety has a different affinity at extracellular pH and at an endosomal pH.

[0181] The term “engineered,” when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences and is in a form suitable for use within genetically engineered protein production systems. Such engineered molecules are those that are separated from their natural environment and include cDNA and genomic clones (i.e., a prokaryotic or eukaryotic cell with a vector containing a fragment of DNA from a different organism). Engineered DNA molecules of the present invention are free of other genes with which they are ordinarily associated but can include naturally occurring or non-naturally occurring 5 ’and 3’ untranslated regions such as enhancers, promoters, and terminators. [0182] An “engineered” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the engineered polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, e.g., greater than 90% pure, greater than 95% pure, more preferably greater than 98% pure or greater than 99% pure. When used in this context, the term “engineered” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers, heterodimers and multimers, heteromultimers, or alternatively glycosylated, carboxylated, modified, or derivatized forms.

[0183] An “engineered” peptide or protein is a polypeptide that is distinct from a naturally occurring polypeptide structure, sequence, or composition. Engineered peptides include non- naturally occurring, artificial, isolated, synthetic, designed, modified, or recombinantly expressed peptides. Provided herein are engineered TfR-binding peptides, variants, or fragments thereof. These engineered TfR-binding peptides can be further linked to a target-binding moiety or a half-life extending moiety, or can be further linked to an active agent or detectable agent, or any combination of the foregoing.

[0184] Polypeptides of the disclosure include polypeptides that have been modified in any way, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, (5) alter binding affinity at certain pH values, and (6) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) are made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A “conservative amino acid substitution” can refer to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that can be conservative substitutions for one another: i) Alanine (A), Serine (S), and Threonine (T); ii) Aspartic acid (D) and Glutamic acid (E); iii) Asparagine (N) and Glutamine (Q); iv) Arginine (R) and Lysine (K); v) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); vi) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). In some embodiments, a conserved amino acid substitution can comprise a non-natural amino acid. For example, substitution of an amino acid for a non-natural derivative of the same amino acid can be a conserved substitution.

[0185] The terms “polypeptide fragment” and “truncated polypeptide” as used herein can refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length peptide or protein. In various embodiments, fragments are at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length. In various embodiments, fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids in length. A fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0186] As used herein, the terms “peptide” or “polypeptide” in conjunction with “variant”, “mutant”, or “enriched mutant”, or “permuted enriched mutant” can refer to a peptide or polypeptide that can comprise an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Variants of the present disclosure include peptide conjugates or fusion molecules (e.g., peptide constructs or peptide complexes).

[0187] A “derivative” of a peptide or polypeptide can be a peptide or polypeptide that can have been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. [0188] The term “% sequence identity” can be used interchangeably herein with the term “% identity” and can refer to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm, and means that a given sequence is at least 80% identical to another length of another sequence. In various embodiments, the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% or more up to 100% sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%. [0189] The terms “% sequence homology” or “percent sequence homology” or “percent sequence identity” can be used interchangeably herein with the terms “% homology,” “% sequence identity,” or “% identity” and can refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. In various embodiments, the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more up to 100% sequence homology to a given sequence. In various embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0190] A protein or polypeptide can be “substantially pure,” “substantially homogeneous”, or “substantially purified” when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein can typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and e.g., will be over 98% or 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution is provided by using high-pressure liquid chromatography (e.g., HPLC) or other high-resolution analytical techniques (e.g., LC-mass spectrometry).

[0191] As used herein, the term “pharmaceutical composition” can generally refer to a composition suitable for pharmaceutical use in a subject such as an animal (e.g., human or mouse). A pharmaceutical composition can comprise a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. The term “pharmacologically effective amount” can refer to that amount of an agent effective to produce the intended biological or pharmacological result.

[0192] As used herein, the term “pharmaceutically acceptable carrier” can refer to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, or a buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. A “pharmaceutically acceptable salt” can be a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines. [0193] As used herein, the terms “treat”, “treating” and “treatment” can refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition, for example, means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition.

Further, references herein to “treatment” can include references to curative, palliative, and prophylactic or diagnostic treatment.

[0194] Generally, a cell of the present disclosure can be a eukaryotic cell or a prokaryotic cell. A cell can be an epithelial cell. A cell can be a microorganism, bacterial, yeast, fungal or algae cell. A cell can be an animal cell or a plant cell. An animal cell can include a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. A mammalian cell can be obtained from a primate, ape, equine, bovine, porcine, canine, feline, or rodent. A mammal can be a primate, ape, dog, cat, rabbit, ferret, or the like. A rodent can be a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. A bird cell can be from a canary, parakeet, or parrots. A reptile cell can be from a turtles, lizard, or snake. A fish cell can be from a tropical fish. For example, the fish cell can be from a zebrafish (e.g., Danino rerid). A worm cell can be from a nematode (e.g., C. elegans). An amphibian cell can be from a frog. An arthropod cell can be from a tarantula or hermit crab.

[0195] A mammalian cell can also include cells obtained from a primate (e.g., a human or a non-human primate). A mammalian cell can include a blood cell, a stem cell, an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, or an immune system cell.

[0196] As used herein, the term “vector,” generally refers to a DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector. [0197] As used herein, the term “subject,” generally refers to a human or to another animal. A subject can be of any age, for example, a subject can be prenatal, newborn, an infant, a toddler, a child, a pre-adolescent, an adolescent, an adult, or an elderly individual.

[0198] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are in relation to the other endpoint, and independently of the other endpoint. The term “about” as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 can include a range from 8.5 to 11.5.

Peptides

[0199] The selective depletion complexes of the present disclosure can comprise one or more peptides. For example, a selective depletion complex of the present disclosure can comprise a receptor-binding peptide (e.g., a TfR-binding peptide or a PD-L1 -binding peptide) and a targetbinding peptide (e.g., a target-binding EGF variant). In some embodiments, two or more peptides can be connected via a linker. The peptides of the present disclosure (e.g., a TfR- binding peptide, a PD-L1 -binding peptide, an EGFR target-binding peptide, or a peptide comprising a TfR-binding peptide linked to an EGFR target-binding peptide) can be used in a method of selectively depleting a target molecule. The peptides of the present disclosure (e.g., TfR-binding peptide, an EGFR target-binding peptide, or a peptide comprising a TfR-binding peptide linked to an EGFR target-binding peptide) can be recycled to the cell surface following endocytosis.

[0200] In some instances, a peptide as disclosed herein can contain only one lysine residue, or no lysine residues. In some instances, one or more or all of the lysine residues in the peptide are replaced with arginine residues. In some instances, one or more or all of the methionine residues in the peptide are replaced by leucine or isoleucine. One or more or all of the tryptophan residues in the peptide can be replaced by phenylalanine or tyrosine. In some instances, one or more or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, one or more or all of the aspartic acid residues can be replaced by glutamic acid residues. In some instances, one or more or all of the lysine residues in the peptide are replaced by alanine or arginine. In some embodiments, the N-terminus of the peptide is blocked or protected, such as by an acetyl group or a te/7-butyloxycarbonyl group. Alternatively or in combination, the C-terminus of the peptide can be blocked or protected, such as by an amide group or by the formation of an ester (e.g., a butyl or a benzyl ester). In some embodiments, the peptide is modified by methylation on free amines. For example, full methylation is accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.

[0201] In some embodiments, the peptide comprises an amino acid sequence SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. In some embodiments, a dipeptide GS can be added as the first two N-terminal amino acids, for example as shown in SEQ ID NO: 1 - SEQ ID NO: 64, or such N-terminal dipeptide GS can be absent as shown in SEQ ID NO: 96 and SEQ ID NO: 65 - SEQ ID NO: 128 or can be substituted by any other one or two amino acids. In some embodiments, a dipeptide comprising GS is used as a linker or used to couple to a linker to form a peptide conjugate or fusion molecules such as a peptide construct or peptide complex. Similarly, in some embodiments, the peptide comprising the amino acid sequence of any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241 can also comprise a dipeptide comprising GS used as a linker or used to couple to a linker to form a peptide conjugate or fusion molecules such as a peptide construct or peptide complex. In some embodiments, the linker comprises a G_xS_y (SEQ ID NO: 130) peptide, wherein x and y independently are any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and the G and S residues are arranged in any order. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 131), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 132), GGGGG (SEQ ID NO: 133), GSGSGSGS (SEQ ID NO: 134), GSGG (SEQ ID NO: 135), GGGGS (SEQ ID NO: 136), GGGS (SEQ ID NO: 129), GGS (SEQ ID NO: 137), GGGSGGGSGGGS (SEQ ID NO: 138), or a variant or fragment thereof or any number of repeats and combinations thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 139) from DkTx, and EPKSSDKTHT (SEQ ID NO: 140) from human IgG3 can be used as a peptide linker or any number of repeats and combinations thereof. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 141) or a variant or fragment thereof or any number of repeats and combinations thereof. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. The length of the linker can be tailored to maximize binding of the selective delivery complex to both TfR and the target molecule (e.g., EGFR) at the same time including accounting for steric access. In some embodiments, the linker between the TfR- binding and target-binding peptides (e.g., EGFR-b inding peptides) within the selective depletion complex is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 residues incrementally up to 100 residues long, particularly for example if the target molecule is not a soluble protein but rather a cell surface protein or cell receptor protein.

[0202] In some embodiments of the present disclosure, a peptide or peptide complex as described herein comprises a TfR-b inding peptide comprising an amino acid sequence set forth in any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. A TfR-binding peptide as disclosed herein can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 that is at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 residues long, wherein the peptide fragment is selected from any portion of the peptide. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids, for example, confer a particular in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.

[0203] In some instances, the peptides as described herein that are capable of targeting and binding to a TfR comprise no more than 80 amino acids in length, or no more than 70, no more than 60, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, or no more than 10 amino acids in length. In some instances, the peptides as described herein that are capable of targeting and binding to a target molecule comprise no more than 80 amino acids in length, or no more than 70, no more than 60, no more than 50, no more than 40, no more than 35, no more than 30, no more than 25, no more than 24, no more than 23, no more than 22, no more than 21, no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, or no more than 10 amino acids in length.

[0204] In other embodiments, peptides can be conjugated to, linked to, or fused to a carrier or a molecule with targeting or homing function for a cell of interest or a target cell. In other embodiments, peptides can be conjugated to, linked to, or fused to a molecule that extends halflife or modifies the pharmacodynamic and/or pharmacokinetic properties of the peptides, or any combination thereof.

[0205] In some instances, a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof. In some instances, one or more lysine residues in the peptide are replaced with arginine residues. In some embodiments, peptides comprise one or more Arg patches. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more Arg or Lys residues are solvent exposed on a peptide. In some instances, a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 histidine residues.

[0206] The peptides of the present disclosure can further comprise neutral amino acid residues. In some embodiments, the peptide has 35 or fewer neutral amino acid residues. In other embodiments, the peptide has 81 or fewer neutral amino acid residues, 70 or fewer neutral amino acid residues, 60 or fewer neutral amino acid residues, 50 or fewer neutral amino acid residues, 40 or fewer neutral amino acid residues, 36 or fewer neutral amino acid residues, 33 or fewer neutral amino acid residues, 30 or fewer neutral amino acid residues, 25 or fewer neutral amino acid residues, or 10 or fewer neutral amino acid residues.

[0207] The peptides of the present disclosure can further comprise negative amino acid residues. In some embodiments the peptide has 6 or fewer negative amino acid residues, 5 or fewer negative amino acid residues, 4 or fewer negative amino acid residues, 3 or fewer negative amino acid residues, 2 or fewer negative amino acid residues, or 1 or fewer negative amino acid residues. While negative amino acid residues can be selected from any negatively charged amino acid residues, in some embodiments, the negative amino acid residues are either E, or D, or a combination of both E and D.

[0208] In some embodiments of the present disclosure, a three-dimensional or tertiary structure of a peptide is primarily comprised of beta-sheets and/or alpha-helix structures. In some embodiments, designed or engineered TfR-b inding peptides or target-binding of the present disclosure are small, compact peptides or polypeptides stabilized by intra-chain disulfide bonds (e.g., mediated by cysteines) to form cystine and a hydrophobic core. In some embodiments, engineered TfR-b inding peptides have structures comprising helical bundles with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptides. In other embodiments, the engineered TfR-b inding peptides or target-binding peptides comprise structures with three alpha helices and three intra-chain disulfide bonds, one between each of the three alpha helices in the bundle of alpha helices.

Receptor-Binding Peptides

[0209] Disclosed herein are peptide sequences, such as those listed in TABLE 1 and TABLE 2, capable of binding to a receptor (e.g., a transferrin receptor or PD-L1 (also known as programmed death-ligand 1)). The peptide capable of binding a receptor may be referred to as a receptor-binding peptide. In some embodiments, a receptor-binding peptide may bind to a recycled receptor that undergoes recycling via a recycling pathway. The depletion of a selected target (e.g., EGFR) by the SDCs described herein is dependent on the normal trafficking and cycling behavior of the recycling receptor in cells to which the recycling receptor-binding peptide the in the SDC is bound. The recycled receptor may be endocytosed into an early endosome and packaged into a recycling endosome prior to maturation of the early endosome into a late endosome. The recycling endosome containing the recycled receptor may fuse with a cell membrane and return the recycled receptor to the cell surface. In some embodiments, a receptor-binding peptide of the present disclosure may remain bound to the receptor during the recycling process, thereby recycling the receptor-binding peptide as well. Examples of recycled receptors that may be targeted by a receptor-binding peptide include transferrin receptor, programmed death-ligand 1, cation-independent mannose 6 phosphate receptor (CI-M6PR), asialoglycoprotein receptor (ASGPR), CXCR7, folate receptor, or Fc receptors (including but not limited to neonatal Fc receptor (FcRn) or FcyRIIb). In some embodiments, a receptorbinding peptide of the present disclosure may comprise a miniprotein, a nanobody, an antibody, an IgG, an antibody fragment, a Fab, a F(ab)2, an scFv, an (scFv)2, a DARPin, or an affibody. In some embodiments, receptor binding can be achieved by engineering an Fc domain for improved binding to an existing Fc receptor, e.g., FcRn or FcyRIIb, or for novel binding to a non-native receptor, e.g., TfR. In some embodiments, the receptor-binding peptide may comprise a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some embodiments, receptor binding can be achieved by conjugation of a target-binding peptide or peptide complex with a sugar or other small molecule that is bound by the cellular receptor (e.g., mannose-6-phosphonate or N-acetylgalactosamine that bind with CI-M6PR and ASGPR, respectively).

[0210] In some embodiments, a receptor-binding peptide of the present disclosure can bind to the receptor (e.g., a recycled receptor) with an affinity that is pH-independent. For example, a receptor-binding peptide can bind the receptor at an extracellular pH (about pH 7.4) with an affinity that is substantially the same the binding affinity at an endocytic pH (such as about pH 5.5 or about pH 6.5). In some embodiments, a receptor-binding peptide can bind the receptor at an extracellular pH (about pH 7.4) with an affinity that is lower than the binding affinity at an endocytic pH (such as about pH 5.5 or about pH 6.5). In some embodiments, a receptor-binding peptide can bind the receptor at an extracellular pH (about pH 7.4) with an affinity that is higher than the binding affinity at an endocytic pH (such as about pH 5.5 or about pH 6.5). In some embodiments, the binding affinity of a receptor-binding peptide for the receptor at extracellular pH (about pH 7.4) and the binding affinity of a receptor-binding peptide for the receptor at endocytic pH (about pH 5.5) can differ by no more than about 1%, no more than about 2%, no more than about 3%, no more than about 4%, no more than about 5%, no more than about 6%, no more than about 7%, no more than about 8%, no more than about 9%, no more than about 10%, no more than about 12%, no more than about 15%, no more than about 17%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, or no more than about 50%. In some embodiments, the affinity of the receptor-binding peptide for the receptor at pH 7.4 and at pH 5.5 can differ by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15 -fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40- fold, or no more than 50-fold. In some embodiments, a receptor-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, SEQ ID NO: 1 - SEQ ID NO: 64, SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241) can be modified to remove one or more histidine amino acids in the receptor-binding interface, thereby reducing the pH-dependence of the binding affinity of the receptor-binding peptide for the receptor. In some embodiments, a receptor-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, SEQ ID NO: 1 - SEQ ID NO: 64, SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241) can lack histidine amino acids in the receptor-binding interface.

[0211] In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at extracellular pH (about pH 7.4). In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at endosomal pH (about pH 5.5). In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at endosomal pH (about pH 5.8).

[0212] In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some embodiments, a receptor-binding peptide with pH- independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. In some embodiments, a receptorbinding peptide with pH-independent binding can bind to the receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.8.

[0213] In some embodiments, the affinity of the receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the affinity of the receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0214] In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with a dissociation rate constant (k_off or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 7.4. In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’ no more than 5xl0’⁴ s’¹, or no more than 2xl0’⁴ s’¹ at pH 5.5. In some embodiments, a receptor-binding peptide with pH-independent binding can bind to the receptor with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.8. [0215] In some embodiments, the dissociation rate constant (k_off or kd) of the receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the dissociation rate constant (koff or kd) of the receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0216] In some embodiments, the receptor-binding peptide can bind to the receptor with an affinity that is pH-dependent. For example, the receptor-binding molecule can bind to the receptor with higher affinity at extracellular pH (about pH 7.4) and with lower affinity at endosomal pH (about pH 5.5), thereby releasing the selective depletion complex from receptor upon internalization and acidification of the endosomal compartment.

[0217] In some embodiments, the recycling receptor may be TfR. A peptide capable of binding transferrin receptor (TfR) may bind TfR or any of the known TfR homologs, including TfRl, TfR2, soluble TfR, or any combination or fragment (e.g., ectodomain) thereof. A peptide capable of binding a transferrin receptor or a TfR homolog can be referred to herein as a transferrin receptor-binding peptide or a TfR-binding peptide. In some embodiments, peptides disclosed herein can penetrate, cross, or enter target cells in a TfR-mediated manner. These cell layers or cells can include TfR-expressing endothelial cells, epithelial cells, and TfR-expressing cells of various tissues or organs such as tumor cells, brain cells, cancerous or tumor cells, liver cells (e.g., hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs), or liver sinusoidal endothelial cells (LSECs)), pancreas cells, colon cells, ovarian cells, breast cells, spleen cells, bone marrow cells, and/or lung cells, or any combination thereof. In some embodiments, a TfR-binding peptide of the present disclosure may comprise a miniprotein, a nanobody, an antibody, an IgG, an antibody fragment, a Fab, a F(ab)2, an scFv, an (scFv)2, a DARPin, or an affibody. In some embodiments, the TfR-binding peptide may comprise a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide.

[0218] In some embodiments, the peptides as discloses herein can cross cellular layers or barriers (e.g., BBB) or cell membranes via, for example, TfR-mediated vesicular transcytosis and TfR-mediated endocytosis, respectively. In addition to binding TfR and promote transcytosis and/or endocytosis, the peptides of the present disclosure can also bind to additional target proteins on cells such as cancer cells. In some cases, a peptide is a peptide or peptide complex comprising a TfR-binding peptide conjugated to, linked to, or fused to a targeting moiety or an active agent (e.g., a therapeutic or diagnostic agent) such as a small molecule or a peptide that has an affinity for an additional target protein (e.g., receptor or enzyme). In some cases, the TfR-binding peptide is linked to a target-binding peptide and enables or promotes TfR-mediated transcytosis of the target-binding peptide across the BBB or TfR-mediated endocytosis into a cell. In some instances, and subsequent to transcytosis, a peptide complex comprising the TfR-binding peptide and a target-binding peptide can target a specific cell or tissue in the CNS and exert a biological effect (e.g., binding a target protein) upon reaching said cell or tissue. In some cases, a peptide complex of the present disclosure exerts a biological effect that is mediated by the TfR-binding peptide, the target-binding peptide, an active agent, or a combination thereof. In some cases, a TfR-binding peptide complex of the present disclosure comprising one target-binding peptides can transport and/or deliver target molecules into cells that express TfR (e.g., deliver target molecules into endosomes). In some cases, the TfR-binding peptide accumulates in tissues in the CNS. In some cases, off-target effects are reduced due to CNS-specific accumulation. In some cases, the TfR-binding peptide accumulates in tissue outside of the CNS (e.g., liver, kidney, spleen, or skin). In some cases, the cells expressing TfR are tumor cells and the TfR-binding peptide complex delivers anti-tumor agents to these tumor cells. In some cases, the anti-tumor agents alone show no or only very limited therapeutic efficacy against the tumor cells; however, when the anti -tumor agents are combined with the TfR-binding peptides of the present disclosure as, for example, a peptide complex, the therapeutic efficacy of these anti-tumor agents is significantly improved.

[0219] In some embodiments, the TfR-binding peptides of the present disclosure (e.g., SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, and SEQ ID NO: 1 - SEQ ID NO: 64) can induce a biologically relevant response. For example, a TfR-binding peptide conjugated to a target-binding peptide can selectively deplete a soluble target molecule or a cell surface target molecule. In some embodiments, the biologically relevant response can be induced after intravenous, subcutaneous, peritoneal, intracranial, or intramuscular dose, and in some embodiments, after a single intravenous, subcutaneous, peritoneal, intracranial, or intramuscular dose. In some embodiments, the TfR-binding peptides can be used in combination with various other classes of therapeutic compounds used to treat and/or prevent pain, neuropathic pain or other neurological disorders such as neurodegenerative disorders, infectious diseases, immunological disorders (e.g., autoimmune diseases) or lysosomal storage diseases. Binding of the herein described peptides and peptide complexes (e.g., peptide conjugates, fusion peptides, or recombinantly produced peptide complexes) to TfR and subsequent transport across a cell layer or barrier such as the BBB (e.g., via TfR-mediated vesicular transcytosis) or a cell membrane (e.g., via TfR-mediated endocytosis) can have implications in a number of diseases, conditions, or disorders associated with over-expression or accumulation of a target molecule (e.g., cancer, neurodegeneration, or lysosomal storage diseases) or diseases associated with mutations (e.g., mutations causing constitutive activity, resistance to treatment, or dominant negative activity) in soluble or surface proteins in a subject (e.g., a human).

[0220] Binding of the herein described peptides and peptide complexes (e.g., peptide conjugates, fusion peptides, or recombinantly produced peptide complexes) to TfR and subsequent transport across a cell layer or barrier such as the BBB (e.g., via vesicular transcytosis) or a cell membrane (e.g., via endocytosis) can have implications in a number of diseases, conditions, or disorders associated with EGFR-driven diseases in the CNS, such as EGFR-driven cancers.

[0221] In some embodiments, TfR-binding peptides of the present disclosure can bind to any of the known TfR homologs, including TfRl, TfR2, soluble TfR, or any combination or fragment (e.g., ectodomain) thereof. Thus, as used herein, “TfR” can refer to any known homolog, derivative, fragment, or member of the TfR family including TfRl, TfR2, and a soluble TfR. In other embodiments, peptides are capable of binding to one, one or more, or all TfR homologs. In some embodiments, peptides of the present disclosure can bind to a TfR and promote a particular biological effect such as vesicular transcytosis. In some embodiments, TfR-binding peptides of the present disclosure, including peptides and peptide complexes with amino acid sequences set forth in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, and SEQ ID NO: 1 - SEQ ID NO: 64, and any derivatives or variant thereof, prevent or decrease the binding of endogenous TfR binders (e.g., transferrin or any derivatives such as apo-transferrin or holo-transferrin) to TfR. In some embodiments, peptides or peptide complexes of the present disclosure comprise derivatives and variants with at least 40% homology, at least 50% homology, at least 60% homology, at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to amino acid sequences set forth in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, and SEQ ID NO: 1 - SEQ ID NO: 64.

[0222] In various embodiments, the interface residues of the TfR-binding peptides of the present disclosure (e.g., those amino acid residues that interact with TfR for receptor binding) can be divided between two largely helical domains of the peptide. In some cases, the interface residues can comprise residues corresponding to residues 5-25 (e.g., and comprising corresponding residues G5, A7, S8, Ml 1, N14, L17, E18, and E21), with reference to SEQ ID NO: 32, or corresponding to residues 35-51 (e.g., and comprising corresponding residues L38, L41, L42, L45, D46, H47, H49, S50, and Q51), with reference to SEQ ID NO: 32, or both. For example, the interface residues can comprise residues corresponding to residues 5-25 (e.g., and comprising corresponding residues G5, A7, S8, Mi l, N14, L17, E18, and E21), with reference to SEQ ID NO: 32, or corresponding to residues 35-51 (e.g., and comprising corresponding residues L38, L41, L42, L45, D46, H47, H49, S50, and Q51), with reference to SEQ ID NO: 32. In some embodiments, a TfR-binding peptide can comprise a fragment of a peptide provided herein, wherein the fragment comprises the minimum interface residues for binding, for example residues corresponding to residues 5-25 (e.g., and comprising corresponding residues G5, A7, S8, Ml 1, N14, L17, E18, and E21), with reference to SEQ ID NO: 32, or corresponding to residues 35-51 (e.g., and comprising corresponding residues L38, L41, L42, L45, D46, H47, H49, S50, and Q51), with reference to SEQ ID NO: 32. In some cases, the TfR-binding peptide is a peptide having the sequence set forth in SEQ ID NO: 32 comprising the TfR-binding residues corresponding to residues G5, A7, S8, Mi l, N14, L17, E18, and E21 of the domain and corresponding to residues L38, L41, L42, L45, D46, H47, H49, S50, and Q51 of the second domain, with reference to SEQ ID NO: 32.

[0223] In some embodiments, TfR-binding peptides bind to TfR with equal, similar, or greater affinity (e.g., lower equilibrium dissociation constant KD) as compared to endogenous molecules (e.g., transferrin, holotransferrin (iron-bound transferrin), apotransferrin (transferrin not bound to iron), or any other endogenous TfR ligands) or other exogenous molecules. In some embodiments, the peptide can have an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM. In some embodiments, the peptide can have an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM. In some embodiments, the peptide can have a dissociation rate constant (k_off or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹. In some embodiments, peptide transport by TfR is improved by having a lower affinity (e.g., a higher equilibrium dissociation constant KD) as compared to endogenous molecules. In some embodiments, peptide transport by TfR is improved by having a faster off rate or higher k_off than endogenous molecules. In some embodiments, the dissociation rate constant (kd or koff) is similar to that of transferrin. In some embodiments, peptide transport is improved by having a faster on rate or a higher k_on, optionally such as higher than that of transferrin. In other embodiments, one or more conserved residues at the transferrin (Tf)-TfR- binding interface are also present in the amino acid sequences of the peptides described herein. In some embodiments, a TfR-binding peptide has an off rate that is slower than the recycling rate of TfR, such that the TfR-binding peptide is likely to remain bound to TfR during the recycling process. In some embodiments, the TfR-binding peptide may have a half-life of dissociation that is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, or no faster than 20 minutes, no faster than 30 minutes, no faster than 45 minutes, no faster than 60 minutes, no faster than 90 minutes, or no faster than 120 minutes. In some embodiments, the TfR-binding peptide may have a half-life of dissociation that is from about 1 minute to about 20 minutes, from about 2 minutes to about 15 minutes, from about 2 minutes to about 10 minutes, or from about 5 minutes to about 10 minutes. In some embodiments, a rate of dissociation of the target-binding peptide from the target molecule is faster than a recycling rate of the cellular receptor. In some embodiments, a half-life of dissociation of the target molecule binding-binding peptide from the target molecule is less than 10 seconds, less than 20 seconds, less than 30 seconds, less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, or less than 60 minutes in endosomal conditions.

[0224] In some embodiments, TfR-binding peptides that exhibit an improved TfR receptor binding show improved transcytosis function, improved endocytosis function, improved recycling, or combinations thereof. In some embodiments, TfR-binding peptides that exhibit an improved TfR receptor binding show no or small changes in transcytosis function, endocytosis function, recycling, or combinations thereof. In some embodiments, TfR-binding peptides that exhibit an improved TfR receptor binding show reduced transcytosis function, reduced endocytosis function, reduced recycling, or combinations thereof. In some embodiments, the TfR-b inding peptide binds at a site of high homology between human and murine TfR, including one or more, or all, of the amino acid domains corresponding to residues 506-510, 523-531, and 611-662 of the human TfR (SEQ ID NO: 190, MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVT KPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPA ARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREF KLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLV HANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNA ELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCP SDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVWGAQRDAW GPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGY LSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWA SKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARA AAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARG DFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSG SHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF). In some embodiments, the regions of TfR to which the peptides disclosed herein or variants thereof bind all or in part to such TfR domains. In some embodiments, the peptides disclosed herein bind to any one, any two, or all three of the TfR regions of high homology including the amino acid domains corresponding to residues 506-510, 523-531, and 611-662 of the human TfR (SEQ ID NO: 190). In some embodiments the peptides disclosed herein bind at least to the domain corresponding to residues 611-662 of the human TfR.

[0225] In some embodiments, the KA and KD values of a TfR-binding peptide can be modulated and optimized (e.g., via amino acid substitutions) to provide an optimal ratio of TfR-binding affinity and efficient transcytosis function.

[0226] In some embodiments, peptides disclosed herein or variants thereof bind to TfR at residues found in the binding interface (e.g., the binding domain or the binding pocket) of TfR with other exogenous or endogenous ligands (e.g., transferrin (Tf), Tf derivatives, or Tf-like peptides or proteins). In some embodiments, a peptide disclosed herein or a variant thereof, which binds to TfR, comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to a sequence that binds residues of TfR, which makeup the binding pocket. In some embodiments, a peptide disclosed herein or a variant thereof, which binds to TfR, comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to an endogenous or exogenous polypeptide known to bind TfR, for example, endogenous Transferrin or any one of the peptides listed in TABLE 1. In other embodiments, a peptide described herein binds to a protein of interest, which comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to TfR, a fragment, homolog, or a variant thereof.

[0227] In some embodiments, peptides disclosed herein or variants thereof bind regions of TfR that comprise the amino acid residues corresponding to residues 506-510, 523-531, and 611-662 (the numbering of these amino acid residues is based on the following Uniprot reference protein sequence of endogenous human TFRC UniProtKB - P02786 (SEQ ID NO: 190, TFR1 HUMAN)). In some embodiments, the regions of TfR to which the peptides disclosed herein or variants thereof bind overlap with those of Tf, a fragment, homolog, or a variant thereof.

[0228] In other embodiments, a nucleic acid, vector, plasmid, or donor DNA comprises a sequence that encodes a peptide, peptide construct, a peptide complex, or variant or functional fragment thereof, as described in the present disclosure. In further embodiments, certain parts or fragments of TfR-binding motifs (e.g., conserved binding motifs) can be grafted onto a peptide or peptide complex with a sequence of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. In some embodiments, peptides can cause TfR to be degraded, prevent TfR from localizing to a cell’s nucleus, or prevent TfR from interacting with transferrin or transferrin-like proteins.

[0229] In some embodiments, a peptide can be selected for further testing or use based upon its ability to bind to the certain amino acid residue or motif of amino acid residues. The certain amino acid residue or motif of amino acid residues in TfR can be identified an amino acid residue or sequence of amino acid residues that are involved in the binding of TfR to Tf. A certain amino acid residue or motif of amino acid residues can be identified from a crystal structure of the TfR:Tf complex. In some embodiments, peptides (e.g., CDPs) demonstrate the resistance to heat, protease (pepsin), and reduction.

[0230] The peptides, peptide complexes (e.g., peptide conjugates or fusion peptides), and selective delivery complexes comprising one or more of the amino acid sequences set forth in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 can bind to a protein of interest. In some embodiments, the protein of interest is a TfR. In some embodiments, the peptides and peptide complexes (e.g., peptide conjugates or fusion peptides) that bind to a TfR comprise at least one of the amino acid sequences set forth in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. In some embodiments, peptides, peptide complexes (e.g., peptide conjugates and fusion molecules) of the present disclosure that bind to a TfR comprise peptide derivatives or variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to amino acid sequences set forth in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. For example, peptides or peptide complexes (e.g., peptide conjugates and fusion molecules) of the present disclosure that bind to a TfR can comprise peptide derivatives or variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to the amino acid sequence set forth in SEQ ID NO: 96. For example, peptides or peptide complexes (e.g., peptide conjugates and fusion molecules) of the present disclosure that bind to a TfR can comprise peptide derivatives or variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to the amino acid sequence set forth in SEQ ID NO: 66. For example, peptides or peptide complexes (e.g., peptide conjugates and fusion molecules) of the present disclosure that bind to a TfR can comprise peptide derivatives or variants having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology or at least 100% homology to the amino acid sequence set forth in SEQ ID NO: 65.

[0231] TABLE 1 lists exemplary peptide sequences according to the methods and compositions of the present disclosure.

TABLE 1 - Exemplary TfR-Binding Peptide Sequences

[0232] In some embodiments, a TfR-binding peptide disclosed herein comprises REGCAX1RCX2KYX4DEX2X3KCX3ARMMSMSNTEEDCEQEX2EDX2X2YCX2X3X5CX5X1 X₄ (SEQ ID NO: 167) or

GSREGCAX1RCX2KYX4DEX2X3KCX3ARMMSMSNTEEDCEQEX2EDX2X2YCX2X3X5CX5 X1X4 (SEQ ID NO: 148), wherein Xi can be independently selected from S, T, D, or N, X2 can be independently selected from A, M, I, L, or V, X3 can be independently selected from D, E, N, Q, S, or T, X₄ can be independently selected from D, E, H, K, R, N, Q, S, or T, and X5 can be independently selected from H, K, R, N, Q, S, or T.

[0233] In some embodiments, a TfR-binding peptide disclosed herein comprises REXICX2X3RCX4KYX5DEX6X₇KCX₈ARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 168) or

GSREX1CX2X3RCX4KYX₅DEX6X7KCX₈ARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 149), wherein Xi, X2, X3, X4, X5, Xe, X7 and X₈ are TfR binding interface residues and can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCMKYNDELEKCEARMMSMSNTEEDCEQEXIEDX2X3YCX4X5X6CX₇X₈X9 (SEQ ID NO: 169) or

GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQEXIEDX2X3YCX4X5X6CX₇X₈X9 (SEQ ID NO: 150), wherein Xi, X2, X3, X4, X5, Xe, X₇, X₈, and X9 are TfR binding interface residues and can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises REX1CX2X3RCX4KYX5DEX6X7KCX8ARMMSMSNTEEDCEQEX9EDX10X11YCX12X13X13C Xi₅Xi₆Xi7 (SEQ ID NO: 170) or GSREX1CX2X3RCX4KYX5DEX6X7KCX8ARMMSMSNTEEDCEQEX9EDX10X11YCX12X13X1 ₃CX1₅X16X17 (SEQ ID NO: 151), wherein Xi, X₂, X3, X₄, X₅, X6, X₇, Xs, X₉, X10, Xn, Xi₂, Xi₃, X14, X15, Xi6 and X17 are TfR binding interface residues and can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 96) or GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 32). In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCTKYNAELEKCEARVMSMSNTEEDCEQELEDLLHCLDHCHSQ (SEQ ID NO: 66) or GSREGCASRCTKYNAELEKCEARVMSMSNTEEDCEQELEDLLHCLDHCHSQ (SEQ ID NO: 2). In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCTKYNAELEKCEARVSSMSNTEETCVQELFDLLHCVDHCVSQ (SEQ ID NO: 65) or GSREGCASRCTKYNAELEKCEARVSSMSNTEETCVQELFDLLHCVDHCVSQ (SEQ ID NO: 1).

[0234] In some embodiments, a TfR-binding peptide disclosed herein comprises X1X2X3X4GX5ASX6X7MX8X9NX10X11LEX12X13EX14X15X16X17X18X19X20X21X22X23X24X25X26 X27X28X29X30X31X32X33X34X35X36X37X38X39X40X41X42X43 (SEQ ID NO: 152), wherein Xi, X₂, X3, X₄, X₅, X₆, X₇, X₈, X₉, X10, Xn, X12, X13, X14,X15, X16, X17, X18, X19,X₂0, X21, X₂2, X23, X₂₄, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, and X43 can independently be any amino acid.

[0235] In some embodiments, a TfR-binding peptide disclosed herein comprises XlX2X3X4X₅X6X7X8X₉XloXl 1X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30 X31X32X33X34X35X36X37LX38X39LLX40X41LDHX42HSQ (SEQ ID NO: 153), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, X11, X12,X13, X14, X15, X16, X17, X18,X19, X₂0, X21,X₂2, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, and X42 can independently be any amino acid.

[0236] In some embodiments, a TfR-binding peptide disclosed herein comprises

X1X2X3X4GX5ASX6X7MX8X9NX10X11LEX12X13EX14X15X16X17X18X19X20X21X22X23X24X25X26

X27X28X29LX30X31LLX32X33LDHX34HSQ (SEQ ID NO: 154), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20,X21, X₂2, X₂3, X₂4, X₂₅, X₂6, X₂7, X₂8, X29, X30, X31, X32, X33, and X34 can independently be any amino acid.

[0237] In some embodiments, a TfR-binding peptide or peptide complex disclosed herein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence homology to any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO:

64, or any variant, homolog, or functional fragment thereof. In some embodiments, a TfR- binding peptide or peptide complex disclosed herein comprises any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or any variant, homolog, or functional fragment thereof. In some embodiments, a peptide that binds to a TfR comprises the amino acid sequence set forth in SEQ ID NO: 96 or SEQ ID NO: 32. In some embodiments, a peptide that binds to a TfR comprises the amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 2. In some embodiments, a peptide that binds to a TfR comprises the amino acid sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 1.

[0238] In some embodiments, a TfR-binding peptide comprises canonical amino acid residues as surface interface residues at any one of the corresponding positions 5, 7, 8, 14, 17, 18, 21, 38, 42, 45, 46, 47, 50, 51, with reference to SEQ ID NO: 32 or a combination thereof. In some embodiments, a TfR-binding peptide comprises canonical amino acid residues as surface interface residues at any one of the corresponding positions G5, A7, S8, N14, L17, E18, E21, L38, L42, L45, D46, H47, S50, Q51, with reference to SEQ ID NO: 32 or a combination thereof. In some embodiments, the peptide or peptide complex of the present disclosure comprises at least one or more of these corresponding residues in SEQ ID NO: 96, SEQ ID NO:

65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. Such peptides can accordingly be engineered with enhanced binding to TfR. In some embodiments, a TfR-binding peptide disclosed herein comprises XlX2X3X4GX₅ASX6X7X₈X9X10NXllX12LEX13X14EXl₅X16X17Xl₈X19X20X21X22X23X24X25X26X₂ 7X2₈X29X3OLX3IX32X33LX34X3₅LDHX36X37SQ (SEQ ID NO: 155), wherein Xi, X2, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, Xu, X12, X13, X14, X15, X16, X17, Xi₈, X19, X20, X21, X22, X23, X₂₄, X₂₅, X₂₆, X27, x₂₈, X29, X30, X31, X32, X33, X34, X35, X36, and X37 can independently be any amino acid. [0239] In some embodiments, surface-distal hydrophilic amino acid residues (e.g., D, E, H, K,

R, N, Q, S, or T) present in the amino acid sequence of a peptide contribute to peptide solubility. In some embodiments, a peptide as disclosed herein comprises a hydrophilic amino acid residue at any one of the corresponding positions 3, 4, 9, 11, 15, 16, 19, 23, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, 40, with reference to SEQ ID NO: 32, or any combination thereof. In some instances, a peptide of the present disclosure comprises hydrophilic amino acid residues at the following corresponding positions: R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, D40, with reference to SEQ ID NO: 32, or any combination thereof. In some embodiments, any one of or any combination of corresponding positions R3, E4, R9, K12, D15, E16, K19, R23, S26, S28, N29, T30, E31, E32, D33, E35, Q36, E37, E39, D40 with reference to SEQ ID NO: 32, can be mutated to another hydrophilic residue without significantly impacting solubility or TfR-binding. In some embodiments, a TfR-binding peptide disclosed herein comprises

X1X2REX3X4X5X6RX7X8KX9X10DEX11X12KX13X14X15RX16X17SX18SNTEEDX19EQEX20EDX 21X22X23X24X25X26X27X28X29X30X31 (SEQ ID NO: 156), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, Xu, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X₂₄, X₂₅, X₂₆, X₂₇, X₂8, X₂₉, X30, and X31 can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises XiX2GCASX3CMX4YNX₅X6LEX7CEAX8MMX9MXioXiiXi2Xi3Xi4Xi₅CXi6Xi7Xi8LXi9X2oL LYCLDHCHSQ (SEQ ID NO: 171) or GSXiX2GCASX3CMX4YNX₅X6LEX7CEAX8MMX9MXioXiiXi2Xi3Xi4Xi₅CXi6Xi7Xi8LXi9X2 oLLYCLDHCHSQ (SEQ ID NO: 157), wherein Xi, X₂, X3, X₄, X₅, X6, X₇, X₈, X₉, Xio, Xn, Xi₂, X13, X14, X15, Xi6, X17, Xis, X19, and X20 can be independently selected from D, E, H, K, R, N, Q,

S, or T.

[0240] In some embodiments, a peptide of the present disclosure comprises cysteine amino acid residues at corresponding positions 4, 8, 18, 32, 42, and 46 with reference to SEQ ID NO: 96. In some embodiments, a peptide of the present disclosure comprises cysteine amino acid residues at corresponding positions 6, 10, 20, 34, 44, and 48 with reference to SEQ ID NO: 32. In some embodiments, a peptide of the present disclosure comprises hydrophilic residues (e.g., D, E, H, K, R, N, Q, S, or T) at corresponding positions 15, 35, 39, 49, with reference to SEQ ID NO: 32, or any combination thereof. In some instances, a peptide of the present disclosure comprises hydrophilic amino acid residues at the following corresponding positions: D15, E35, E39, H49, with reference to SEQ ID NO: 32, or any combination thereof. In some embodiments, any one of or any combination of corresponding positions D15, E35, E39, H49 with reference to SEQ ID NO: 32, can be mutated to another hydrophilic residue without significantly impacting solubility or TfR-binding. In some embodiments, a TfR-binding peptide disclosed herein comprises. In some embodiments, a TfR-binding peptide disclosed herein comprises

XlX2X3X4X₅X6X7X₈X₉XloXl 1X12X13X14DX15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X 30X31X32X33EX34X35X36EX37X38X39X40X41X42X43X44X45HX46X47 (SEQ ID NO: 158), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, Xu, X12, X13, X14, X15, Xi6, X17, Xis, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, and X47 can independently be any amino acid. In some embodiments, a TfR- binding peptide disclosed herein comprises REGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 172) or

GSREGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 159), wherein Xi, X2, X3, and X4 can be independently selected from D, E, H, K, R, N, Q, S, or T.

[0241] In some embodiments, a peptide of the present disclosure comprises hydrophobic residues (e.g., A, M, I, L, V, F, W, or Y) at corresponding positions 15, 35, 39, 49, with reference to SEQ ID NO: 32, or any combination thereof. In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 173) or GSREGCASRCMKYNX1ELEKCEARMMSMSNTEEDCX2QELX3DLLYCLDHCX4SQ (SEQ ID NO: 160), wherein Xi, X2, X3, and X4 can be independently selected from A, M, I, L, V, F, W, or Y. In some embodiments, hydrophilic amino acid residues at any one of the corresponding positions 15, 35, 39, and 49, with reference to SEQ ID NO: 32, are associated with higher binding affinity for TfR (e.g., target engagement) and higher solubility. In some embodiments, mutation of an amino acid residue at any one of the corresponding positions 15, 35, 39, and 49, with reference to SEQ ID NO: 32, from a hydrophobic to a hydrophilic residue can lead to higher binding affinity for TfR (e.g., target engagement) and higher solubility.

[0242] In some embodiments, a peptide of the present disclosure comprises hydrophobic residues (e.g., A, M, I, L, V, F, W, or Y) at corresponding positions 11, 25, 27, with reference to SEQ ID NO: 32, or any combination thereof. In some embodiments, a peptide of the present disclosure comprises hydrophilic residues (e.g., D, E, H, K, R, N, Q, S, or T) at corresponding positions 11, 25, 27, with reference to SEQ ID NO: 32, or any combination thereof. In some embodiments, hydrophobic amino acid residues at any one of the corresponding positions 11 , 25, and 27, with reference to SEQ ID NO: 32, are associated with higher binding affinity for TfR (e.g., target engagement) and higher solubility. In some embodiments, mutation of an amino acid residue at any one of the corresponding positions 11, 25, and 27, with reference to SEQ ID NO: 32, from a hydrophilic residue to a hydrophobic residue can lead to higher binding affinity for TfR (e.g., target engagement) and higher solubility. In some embodiments, a peptide of the present disclosure comprises hydrophobic amino acid residues at the corresponding positions Mi l, M25, M27, with reference to SEQ ID NO: 32, or any combination thereof. In some instances, a peptide comprises the hydrophobic amino acid residues at the corresponding positions Mi l, M25, and M27, with reference to SEQ ID NO: 32. In some embodiments, any combination of the corresponding positions Mi l, M25, and M27, with reference to SEQ ID NO: 32, can be mutated to another hydrophobic residue without significantly impacting solubility or TfR-binding. In some embodiments, a TfR-binding peptide disclosed herein comprises X1X2X3X4X₅X6X7X₈X9X1OMX11X12X13X14X1₅X16X17X1₈X19X2OX21X22X23MX24MX25X26X27X₂8 X29X30X31X32X33X34X35X36X37X3₈X39X40X41X42X43X44X45X46X47X₄₈ (SEQ ID NO: 161), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, Xio, Xu , X12, X13, Xi₄, X15, Xi₆,Xi₇, Xi₈, X19, X20, X21, X22, X23, X24, X25, X26, X27, X2₈, X29, X30, X3I , X32, X33, X34, X35, X36, X37, X3₈, X39, X40, X4I , X42, X43, X44, X45, X46, X47, and X4₈ can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 174) or GSREGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 162), wherein Xi, X₂, and X3 can be independently selected from A, M, I, L, V, F, W, or Y. In some embodiments, a TfR-binding peptide disclosed herein comprises REGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 175) or GSREGCASRCX1KYNDELEKCEARMX2SX3SNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 163), wherein Xi, X2, and X3 can be independently selected from D, E, H, K, R, N, Q, S, or T.

[0243] In some embodiments, a peptide of the present disclosure comprises an aliphatic amino acid residue (e.g., A, M, I, L, or V) at corresponding position 45, with reference to SEQ ID NO: 32. In some embodiments, a peptide of the present disclosure comprises an aromatic amino acid residue (e.g., F, W, or Y) at corresponding position 45, with reference to SEQ ID NO: 32. In some embodiments, an aliphatic amino acid residue at corresponding position 45, with reference to SEQ ID NO: 32, is associated with higher binding affinity to TfR. In some instances, a peptide comprises the aliphatic amino acid residue corresponding to L45, with reference to SEQ ID NO: 32. In some embodiments, mutation of an amino acid residue at corresponding position 45 from an aromatic residue to an aliphatic reside can lead to higher binding affinity for TfR (e.g., target engagement) and higher solubility. In some embodiments, mutating corresponding position L45, with reference to SEQ ID NO: 32, to another aliphatic residue may not significantly impact solubility or TfR-binding. In some embodiments, a TfR-binding peptide disclosed herein comprises

XlX2X3X4X₅X6X7X₈X₉XloXl 1X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30 X31X32X33X34X35X36X37X38X39X40X41X42X43X44LX45X46X47X48X49X50 (SEQ ID NO: 164), wherein Xi, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X10, Xu, X12, X13, Xi₄, X15, Xi₆,Xi₇, Xis, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, and X50 can independently be any amino acid. In some embodiments, a TfR-binding peptide disclosed herein comprises GSREGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCXiDHCHSQ (SEQ ID NO: 165) or REGCASRCMKYNDELEKCEARMMSMSNTEEDCEQELEDLLYCXiDHCHSQ (SEQ ID NO: 176), wherein Xi can be independently selected from A, M, I, L, or V.

[0244] In some embodiments, a peptide of the present disclosure comprises GSREGCASRCMX1YNDELEX2CEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 166) or REGCASRCMX1YNDELEX2CEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ (SEQ ID NO: 177), wherein Xi and X2 can be independently selected from K or R. In some embodiments, these residues at corresponding position 12 and 19, with reference to SEQ ID NO: 32, can be used for chemical conjugation to another molecule (e.g., an active or a detectable agent). In some embodiments, Xi and X2 are both R and chemical conjugation occurs at the N-terminus of the peptide.

[0245] In some embodiments, a receptor-binding peptide may be derived from an antibody or antibody fragment. For example, a receptor-binding peptide may be derived from a single chain antibody fragment (scFv). Examples of TfR-binding peptides that may be incorporated into a selective depletion complex of the present disclosure include SEQ ID NO: 220

(QVQLQESGGGWQPGRSLRLSCAASRFTFSSYAMHWVRQAPGKGLEWVAVISYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGT LVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQK PGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTG PVFGGGTKLTVLG), SEQ ID NO: 221 (QVQLQESGGGWQPGRSLRLSCAASRFTFNNYAMHWVRQAPGKGLEWVAVISYDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQ GTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQ QKPGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSL TGPVFGGGTKLTVLG), and SEQ ID NO: 222 (QVQLQESGGGWQPGRSLRLSCAASRYPFHHHDHHWVRQAPGKGLEWVAVISYDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQ GTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQ QKPGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSL TGPVFGGGTKLTVLG). In some embodiments, a TfR-binding peptide may have a sequence of any one of SEQ ID NO: 220 - SEQ ID NO: 222, or a fragment thereof. In some embodiments, a

TfR-binding peptide may have a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 220 - SEQ ID NO: 222, or a fragment thereof. In some embodiments, a peptide of SEQ ID NO: 220 or SEQ ID NO: 221 may function as a pH- independent TfR-binding peptide. In some embodiments, a peptide of SEQ ID NO: 222 may function as a pH-dependent TfR-binding peptide.

[0246] In some embodiments, mutations in any one or more of the amino acid residues of a peptide of the present disclosure can improve binding affinity of the peptide to TfR. In some embodiments, mutations in 5-80% of amino acid residues of a peptide of the present disclosure improve the binding affinity of the peptide to TfR. In some embodiments, mutations in 1-100%, 5-100%, or 5-50% of amino acid residues of a peptide of the present disclosure improve binding affinity of the peptide to TfR. In some embodiments, mutations in 15-50% of amino acid residues of a peptide of the present disclosure improve binding affinity of the peptide to TfR. In some embodiments, mutations in 15-30% of amino acid residues of a peptide of the present disclosure improve binding affinity of the peptide to TfR. In some embodiments, mutations in 25-30% of amino acid residues of a peptide of the present disclosure improve binding affinity of the peptide to TfR. For example, mutations in 14 of the 51 amino acid residues (27.5%) of a peptide having a sequence of SEQ ID NO: 32 can improve binding affinity of the peptide to TfR.

[0247] In some embodiments, mutations in any one or more of the amino acid residues of a peptide of the present disclosure can lie at the binding interface of TfR. In some embodiments, a mutation to a peptide can improve binding affinity, which can be beneficial to binding and transcytosis of a peptide or peptide complex disclosed herein. In some embodiments, the peptides provided herein can have many mutations or few mutations to obtain optimal activity, wherein optimal activity is sufficient binding for engagement of the TfR, but not necessarily binding that is so strong as to preclude release of the peptide and/or peptide complex after transcytosis. Thus, peptides of the present disclosure can comprise a number of mutations (also referred to as % mutated amino acid residues) that tune binding affinity and off rate to obtain optimal binding, function (e.g., transcytosis, BBB-penetration, cell membrane penetration, transport across a biological barrier, endocytosis, recycling, or combinations thereof), and release of the peptide or peptide complex. Thus, mutations that result in the highest possible affinity may not necessarily correlate to a superior peptide having optimal binding and transcytosis.

[0248] In some embodiments, 1-100% or 5-100% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. In some embodiments, 10-90% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. In some embodiments, 20-80% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. In some embodiments, 30-70% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. In some embodiments, 40-60% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. In some embodiments, 30-35% of amino acid residues of a peptide of the present disclosure lie at the binding interface of TfR. For example, 17 of the 51 amino acid residues (33%) of a peptide having a sequence of SEQ ID NO: 32 can lie at the binding interface of TfR.

[0249] In some embodiments, mutations in any one or more of the amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR can improve binding affinity of the peptide to TfR. In some embodiments, mutations in 1-100% or 5-100% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 5-80% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 10-70% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 15-60% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 20-50% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 25-30% of amino acid residues of a peptide of the present disclosure that lie at the binding interface of TfR improve binding affinity of the peptide to TfR. For example, mutations in 5 of the 17 amino acid residues (29%) of a peptide having a sequence of SEQ ID NO: 32 that lie at the binding interface of TfR and can improve binding affinity of the peptide to TfR.

[0250] In some embodiments, mutations in any one or more of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 1-100% or 5-100% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 10-90% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 20-80% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 30-70% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 40- 60% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. In some embodiments, 65-70% of amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR. For example, 34 of the 51 amino acid residues (66%) of a peptide having a sequence of SEQ ID NO: 32 can lie at the binding interface of TfR.

[0251] In some embodiments, mutations in any one or more of the amino acid residues of a peptide of the present disclosure are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 1-100% or 5-100% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 5-80% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 10- 70% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 15-60% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 20-50% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. In some embodiments, mutations in 25-30% of amino acid residues of a peptide of the present disclosure that are distal to the binding interface of TfR improve binding affinity of the peptide to TfR. For example, mutations in 5 of the 17 amino acid residues that are distal to the binding interface of TfR can improve binding affinity of the peptide to TfR. For example, mutations in 9 of the 34 amino acid residues (26.5%) of a peptide having a sequence of SEQ ID NO: 32 that are distal to the binding interface of TfR can improve binding affinity of the peptide to TfR. In some embodiments, and without being bound to any theory, one or more mutations in the amino acid residues of the peptide that are distal to the binding interface of TfR can improve protein folding, enhance protein solubility, and/or alter the backbone geometry that can improve binding through an optimized interface shape complementarity.

[0252] It is understood that for any of the foregoing peptide or peptide complex of the present disclosure describing the mutations and amino acid substitutions and revisions with reference to SEQ ID NO: 32, that the mutations and amino acid substitutions comprise at least one or more of the corresponding residues in SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64.

[0253] In some embodiments, a receptor-binding peptide of the present disclosure may be a PD- L1 -binding peptide. The PD-L1 -binding peptide may be incorporated into a selective depletion complex of the present disclosure to facilitate selective depletion of a target molecule via PD- Ll-mediated endocytosis. In some embodiments, the PD-Ll-binding peptide that is a receptorbinding peptide may bind PD-L1 with an affinity that is pH-independent (for example, a similar affinity at extracellular pH and at an endosomal pH) or may bind PD-L1 with an affinity that is pH-dependent (for example, a higher affinity at extracellular pH and a lower affinity at an endosomal pH). Examples of PD-Ll-binding peptides are provided in TABLE 2.

TABLE 2 - Exemplary PD-Ll-Binding Peptides

[0254] In some embodiments, a PD-L1 -binding peptide disclosed herein comprises a sequence of X¹X²X³CX⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵CX¹⁶X¹⁷X¹⁸X¹⁹X²⁰X²¹X²²X²³X²⁴X²⁵X²⁶X²⁷X²⁸C X²⁹X³⁰X³¹X³²X³³X³⁴X³⁵X³⁶X³⁷CX³⁸X³⁹X⁴⁰CX⁴¹X⁴²X⁴³ (SEQ ID NO: 392), wherein X¹ can independently be selected from E, M, V, or W; X² can independently be selected from G, E, L, or F; X³ can independently be selected from D, E, or S; X⁴ can independently be selected from K, R, or V; X⁵ can independently be selected from E, Q, S, M, L, or V; X⁶ can independently be selected from D, E, H, K, R, N, Q, S, or Y; X⁷ can independently be selected from D, M, or V; X⁸ can independently be selected from A, K, R, Q, S, or T; X⁹ can independently be selected from A, D, E, H, Q, S, T, M, I, L, V, or W; X¹⁰ can independently be selected from A, E, R, Q, S, T, W, or P; X¹¹ can independently be selected from A, E, K, R, N, Q, T, M, I, L, V, or W; X¹² can independently be selected from G, A, E, K, N, T, or Y; X¹³ can independently be selected from G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y, or P; X¹⁴ can independently be selected from D, K, R, N, L, or V; X¹⁵ can independently be selected from G, A, D, T, L, W, or P; X¹⁶ can independently be selected from G, A, E, H, K, N, S, F, or P; X¹⁷ can independently be selected from G, A, D, E, N, or P; X¹⁸ can independently be selected from G, D, H, K, R, N, Q, S, T, V, or Y; X¹⁹ can independently be selected from G, D, E, H, K, N, Q, S, T, M, I, F, W, Y, or P; X²⁰ can independently be selected from G, A, D, E, H, K, R, N, Q, S, Y, or P; X²¹ can independently be selected from G, A, D, H, N, Q, S, V, F, or P; X²² can independently be selected from A, D, H, N, Q, S, T, M, I, V, Y, or P; X²³ can independently be selected from G, A, D, K, R, T, W, or Y; X²⁴ can independently be selected from G, A, E, N, Q, T, I, V, or P; X²⁵ can independently be selected from G, D, N, Q, T, L, V, F, or P; X²⁶ can independently be selected from G, A, E, K, R, N, Q, S, T, I, Y, or P; X²⁷ can independently be selected from A, D, N, or I; X²⁸ can independently be selected from G, D, E, H, N, F, or W; X²⁹ can independently be selected from G, A, E, N, S, Y, or P; X³⁰ can independently be selected from G, M, or L; X³¹ can independently be selected from G, A, D, K, N, Q, or W; X³² can independently be selected from D, E, H, K, N, Q, S, T, L, V, F, Y, or P; X³³ can independently be selected from G, E, Q, or F; X³⁴ can independently be selected from D or K; X³⁵ can independently be selected from G, V, or P; X³⁶ can independently be selected from G, H, R, V, F, W, or P; X³⁷ can independently be selected from A, D, or K; X³⁸ can independently be selected from E, H, Q, L, or F; X³⁹ can independently be selected from D, E, R, S, T, M, L, or F; X⁴⁰ can independently be selected from G, A, D, E, H, K, R, M, L, or P; X⁴¹ can independently be selected from G, A, K, S, I, or L; X⁴² can independently be selected from G, A, D, E, R, Q, T, or F; and X⁴³ can independently be selected from A, H, N, Q, S, F, or P.

[0255] In some embodiments, a binding peptide disclosed herein comprises a sequence of EEDCKVX¹CVX¹X¹X¹X¹X²X³KX¹CX¹EX¹X⁴X¹X¹X¹X¹X¹X¹X¹AX¹CX¹GX¹X⁵FX⁶VFX⁶CLX 'X'CX'X'X¹ (SEQ ID NO: 393), wherein X¹ can independently be selected from any noncysteine amino acid; X² can independently be selected from M, I, L, or V; X³ can independently be selected from Y, A, H, K, R, N, Q, S, or T; X⁴ can independently be selected from D, E, N, Q, or P; X⁵ can independently be selected from K or P; and X⁶ can independently be selected from D or K. [0256] A PD-L1 -binding peptide may comprise a PD-L1 -binding motif that forms part or all of a binding interface with PD-L1. One or more residues of a PD-L1 -binding motif may interact with one or more residues of PD-L1 at the binding interface between the PD-L1 -binding peptide and PD-L1. In some embodiments, multiple PD-L1 -binding motifs may be present in a PD-L1- binding peptide. A PD-L1 -binding motif may comprise a sequence of CX¹X²X³CX⁴X⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²C (SEQ ID NO: 394), wherein X¹ can independently be selected from K, R, or V; X² can independently be selected from E, Q, S, M, L, or V; X³ can independently be selected from D, E, H, K, R, N, Q, S, or Y; X⁴ can independently be selected from D, M, or V; X⁵ can independently be selected from A, K, R, Q, S, or T; X⁶ can independently be selected from A, D, E, H, Q, S, T, M, I, L, V, or W; X⁷ can independently be selected from A, E, R, Q, S, T, W, or P; X⁸ can independently be selected from A, E, K, R, N, Q, T, M, I, L, V, or W; X⁹ can independently be selected from G, A, E, K, N, T, or Y; X¹⁰ can independently be selected from G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y, or P; X¹¹ can independently be selected from D, K, R, N, L, or V; and X¹² can independently be selected from G, A, D, T, L, W, or P. In some embodiments, a PD-Ll-binding motif may comprise a sequence of CKVX¹CVX¹X¹X¹X¹X²X³KX¹C (SEQ ID NO: 396), wherein X¹ can independently be selected from any non-cysteine amino acid; X² can independently be selected from M, I, L, or V; and X³ can independently be selected from Y, A, H, K, R, N, Q, S, or T. In some embodiments, a PD-Ll-binding motif may comprise a sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 398). In some embodiments, a PD-Ll-binding motif may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity to SEQ ID NO: 398.

[0257] A PD-Ll-binding motif may comprise a sequence of X¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹C (SEQ ID NO: 395), wherein X¹ can independently be selected from D, E, H, K, N, Q, S, T, L, V, F, Y, or P; X² can independently be selected from G, E, Q, or F; X³ can independently be selected from D or K; X⁴ can independently be selected from G, V, or P; X⁵ can independently be selected from G, H, R, V, F, W, or P; X⁶ can independently be selected from A, D, or K; X⁷ can independently be selected from E, H, Q, L, or F; X⁸ can independently be selected from D, E, R, S, T, M, L, or F; and X⁹ can independently be selected from G, A, D, E, H, K, R, M, L, or P. In some embodiments, a PD-Ll-binding motif may comprise a sequence of X¹FX²VFX²CLX³X³C (SEQ ID NO: 397), wherein X¹ can independently be selected from K or P; X² can independently be selected from D or K; and X³ can independently be selected from any noncysteine amino acid. In some embodiments, a PD-Ll-binding motif may comprise a sequence of KFDVFKCLDHC (SEQ ID NO: 399). In some embodiments, a PD-L1 -binding motif may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity to SEQ ID NO: 399.

[0258] A PD-Ll-binding peptide (e.g., any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241, or a pH-independent variant thereof) with high affinity PD-Ll-binding at endosomal pH may be complexed with a target-binding peptide as described herein to form a selective depletion complex for selective depletion of the target molecule. The selective depletion complex can be used to selectively deliver a target molecule across a cellular layer or membrane. For example, the selective depletion complex can be used to selectively deliver the target molecule to an endocytic compartment via PD-L1 -mediated endocytosis. The target molecule can be selectively depleted upon binding to the target-binding peptide of the selective depletion complex and endocytosis via PD-L1 -mediated endocytosis as described.

[0259] Selective depletion of a target molecule using PD-L1 -mediated endocytosis may be used to selectively deplete the target molecule specifically in tissues that express PD-L1. In some embodiments, a selective depletion complex comprising a receptor-binding peptide that binds PD-L1 may be used to selectively deplete a target molecule in a PD-L1 positive cancer, a lung tissue, a pancreatic islet tissue, a lymphoid tissue, an immune cell, a gastrointestinal tissue, a bone marrow tissue, a reproductive tissue, a muscle tissue, an adipose tissue, or any other PD-L1 positive tissue. For example, a selective depletion complex comprising a PD-Ll-binding peptide and an ACE2-binding peptide may be used to selectively deplete ACE2 in lung tissue to prevent a viral infection (e.g., a SARS-CoV-2 infection). In another example, a selective depletion complex comprising a PD-Ll-binding peptide and an HLA-b inding peptide may be used to selectively deplete HLA in pancreatic islet cells to prevent T-cell attack of insulin-expressing cells in type I diabetes.

Target-Binding Peptides

[0260] Peptides, peptide complexes, or selective depletion complexes of the present disclosure can comprise a target-binding peptide (e.g., a target-binding EGF variant). The target-binding peptide can be capable of binding a target molecule (e.g., EGFR). In some embodiments, the target-binding peptide can bind to the target molecule with an affinity that is pH-dependent. For example, the target-binding peptide can bind the target molecule with a higher affinity at an extracellular pH (such as about pH 7.4) than at an endosomal pH (such as about pH 5.5). A target-binding peptide can be conjugated to a receptor-binding peptide of the present disclosure (e.g., a TfR-binding peptide any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 or a PD-Ll-binding peptide of any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241) to form a selective depletion complex. The selective depletion complex can be used to selectively deliver a target molecule across a cellular layer or membrane (e.g., BBB or cell membrane). For example, the selective depletion complex can be used to selectively deliver the target molecule (e.g., EGFR) to an endocytic compartment via receptor-mediated endocytosis (e.g., PD-L1 -mediated endocytosis or TfR-mediated endocytosis). The target molecule (e.g., EGFR) can be selectively depleted upon binding to the target-binding peptide of the selective depletion complex and endocytosis via receptor-mediated endocytosis. The target molecule can be an EGFR protein. Selective depletion of a cell surface molecule (e.g., a receptor such as EGFR) using a selective depletion complex comprising a target-binding peptide that binds to the cell surface molecule can result in a reduction of the cell surface molecule (e.g., a surface exposed protein). The surface exposed protein can be associated with a disease or a condition. In some embodiments, a selective depletion complex of the present disclosure can comprise two or more target-binding peptides to promote dimerization of a target molecule. Promoting dimerization can increase internalization of the target molecule, resulting in selective depletion of the target molecule. For example, a selective depletion complex comprising two copies of a target-binding peptide can promote homodimerization of the target molecule.

[0261] In some embodiments, the EGF variant peptide of the present disclosure may further comprise a miniprotein, a nanobody, an antibody, an IgG, an antibody fragment, a Fab, a F(ab)2, an scFv, an (scFv)2, a DARPin, or an affibody. In some embodiments, the target-binding peptide may comprise a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some embodiments, an EGF variant peptide can further comprise an antibody (e.g., IgG or other antibody), an antibody fragment, (e.g., scFv, scFv2, Fab, F(ab)2, or other antibody fragment), or a nanobody (e.g., a VHH-domain nanobody or VNAR-domain nanobody from camelids or sharks), which can be stable at a low pH.

[0262] In some embodiments, a target-binding peptide of the present disclosure (e.g., an EGFR- binding peptide) can bind to the target molecule (e.g., EGFR) with an affinity that is pH- dependent. For example, the target-binding peptide can bind the target molecule at an extracellular pH (such as about pH 7.4) with an affinity that is higher than the binding affinity at an endocytic pH (such as about pH 7.0, pH 6.5, pH 6.0, pH 5.8, or pH 5.5). In some embodiments, the binding affinity of the target-binding peptide for the target molecule at an extracellular pH (about pH 7.4) can be at least about 1.1 -fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5- fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, at least about 1000-fold, at least about 10,000-fold the binding affinity of the target-binding peptide for the target molecule at an endosomal pH (such as about pH 7.0, pH 6.5, pH 6.0, pH 5.8, pH 5.5, or pH 5.0). In some embodiments, the affinity of the target-binding peptide for the target at pH 6.5 or pH 5.5 is no greater than about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the affinity of the target-binding peptide for the target at pH 7.4. In some embodiments, the affinity of the target-binding peptide for the target at pH 7.4 is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the target-binding peptide for the target molecule at pH 6.5 or pH 5.5 [0263] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at extracellular pH (such as about pH 7.4). In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, at least 20 pM, at least 50 pM, at least 100 pM, at least 500 pM, at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 500 mM, or at least 1 M at endosomal pH (about pH 5.5 or about pH 6.5). In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, at least 20 pM, at least 50 pM, at least 100 pM, at least 500 pM, at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 500 mM, or at least 1 M at endosomal pH (about pH 5.8).

[0264] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 7.4. In some embodiments, a target-binding peptide with pH- dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.8.

[0265] In some embodiments, the affinity of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the affinity of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0266] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some embodiments, a target-binding peptide with pH- dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 6.5.

[0267] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (k_off or kd) of no more than 1x10'¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’ no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5xl0’⁵ s’¹, or no more than 2xl0’⁵ s’¹ at pH of 7.4. In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 1x10’¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5xl0’⁵ s’¹, or no more than 2xl0’⁵ s’¹ at pH 6.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’ or no more than 2xl0’⁴ s’¹ at pH 5.5. In some embodiments, a target-binding peptide with pH- dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 5.8.

[0268] In some embodiments, the dissociation rate constant (koff or kd) of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the dissociation rate constant (koff or kd) of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0269] In some embodiments, the dissociation rate constant (k_off or kd) of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.5 at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.5 than at pH 7.4. In some embodiments, the dissociation rate constant (koff or kd) of the target-binding peptide with pH-dependent binding to the target molecule at pH 7.4 and at pH 5.5 at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.8 than at pH 7.4.

[0270] In some embodiments, the target-binding molecule can release the target molecule upon internalization into an endosomal compartment and acidification of the endosome. Such release the target molecule upon acidification of the endosome can occur at about pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. In some embodiments, release of the target molecule can occur at a pH of from about pH 7.0 to about pH 4.5, from about pH 6.5 to about pH 5.0, or from about pH 6.0 to about pH 5.5 or lower.

[0271] Target-binding peptides with pH-dependent binding affinity can be engineered by selective integration of histidine (His) amino acid residues in the target-binding interface. In some instances, a target-binding peptide with pH-dependent binding affinity comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 histidine residues in the target-binding interface. Since the side chain of histidine is predominantly uncharged at pH between about 6.0 and about 9.2 and predominantly positively charged at pH below about 6.0, selectively inserting or removing His residues in a target-binding peptide can impart pH-dependent binding properties. A target-binding peptide can be stable at low pH (e.g., at endosomal pH).

[0272] In some embodiments, release of the target molecule by the target-binding peptide upon internalization into an endosomal compartment can be affected by differences in the ionic strength between the extracellular physiologic environment and endosomal cellular compartments. In some embodiments, the ionic strength of the endosomal compartment is higher than the ionic strength of the extracellular physiologic environment. Ionic strength, which varies with salt concentration, may depend on the concentrations of various electrolytes in solution, for example hydrogen (H⁺), hydroxide (OH’), hydronium (H₃O⁺), sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), manganese (Mn²⁺), chloride (Cl’), carbonate (CO₃ ²’), cobalt (Co²⁺), phosphate (PCU³'), or nitrate (NO,-). In some embodiments, targetbinding peptides with salt-dependent or ionic strength-dependent binding affinity can be engineered by selective integration of salt labile moieties (e.g., polar or charged amino acid side chains) in the target-binding interface that would enable dissociation of the target-binding molecule in the endosome. For example, the target-binding interface of the target-binding peptide may form one or more polar or charge-charge interactions with the target-binding peptide that can be disrupted as the ionic strength of the environment increases.

[0273] In some instances, a target-binding peptide with a binding affinity dependent on ionic strength (e.g., dependent on hydrogen, hydroxide, hydronium, sodium, potassium, calcium, magnesium, manganese, chloride, carbonate, cobalt, phosphate, and/or nitrate concentration) could dissociate over a range of ionic strengths, for example ionic strengths from about 30 mM to about 1 M. In some embodiments, an ionic strength-dependent target-binding peptide with a binding affinity dependent on ionic strength could dissociate at an ionic strength of from about 50 mM to about from about 50 mM to about 1 M, from about 60 mM to about 950 mM, from about 70 mM to about 900 mM, from about 80 mM to about 850 mM, from about 90 mM to about 800 mM, from about 100 mM to about 750 mM, from about 110 mM to about 700 mM, from about 120 mM to about 650 mM, from about 130 mM to about 600 mM, from about 140 mM to about 550 mM, from about 150 mM to about 500 mM, from about 160 mM to about 450 mM, from about 170 mM to about 400 mM, from about 180 mM to about 350 mM, from about 190 mM to about 300 mM, or from about 200 mM to about 250 mM. In some embodiments, the ionic strength-dependent target-binding peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 polar or charge-charge interactions in the target-binding interface (e.g., the interface between EGFR and the EGFR-b inding peptide).

[0274] A target-binding peptide of the present disclosure may bind to a target (e.g., a target molecule), such as EGFR, EGFRvIII, or TKI-resistant EGFR. The target molecule (e.g., EGFR, EGFRvIII, or TKI-resistant EGFR) may be endocytosed and degraded upon binding to the target-binding peptide of a selective depletion complex. The amino acid sequence of soluble EGFRvIII ectodomain (EGFRvIII) is provided as follows:

LEEKKGNYWTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDS LSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW PENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKNLCY ANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVS RGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPH CVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS (SEQ ID NO: 527). In some embodiments, soluble EGFRvIII (e.g., SEQ ID NO: 527) may also comprise a labelling or affinity tag. In some embodiments, soluble EGFRvIII (e.g., SEQ ID NO: 527) may comprise one or more of a G4S spacer (e.g., SEQ ID NO: 136), a poly-His tag (e.g., SEQ ID NO: 142), a biotinylation tag (e.g., SEQ ID NO: 543), or any combination thereof. In some embodiments, soluble EGFRvIII (e.g., SEQ ID NO: 527) may be in a biotinylated form. In some embodiments, soluble EGFRvIII (e.g., SEQ ID NO: 527) may comprise a biotinylation tag (e.g., SEQ ID NO: 543) and undergo biotinylation by a BirA enzyme.

[0275] A target-binding peptide of the present disclosure may bind to a target molecule, such as a target molecule with clinical relevance. In some embodiments, a target molecule may be a protein that is over-expressed or over-activated in a disease or condition. For example, a target molecule may be an EGFR transmembrane protein involved in oncogenic signaling. The target molecule (e.g., EGFR) may be endocytosed and degraded upon binding to the target-binding peptide of a selective depletion complex.

[0276] In some embodiments, a target molecule may be a transmembrane protein, such as a receptor tyrosine kinase. Examples of receptor tyrosine kinases that may be targeted using a selective depletion complex include EGF receptor, or ErbB. In some embodiments, the receptor tyrosine kinase may be EGFR. Targeting the transmembrane protein using a selective depletion complex may lead to internalization and degradation of the transmembrane protein

[0277] Endocytosis and subsequent degradation of the target molecule may treat (e.g., eliminate, reduce, slow progression of, or treat symptoms of) a disease or condition associated with the target molecule. In some embodiments, the degradation of the target molecule may cause remission in, reduce, ameliorate, or ablate a disease or condition (e.g., a cancer). In some embodiments, the degradation of the target molecule may cause remission in, reduce, ameliorate, or ablate a cancer. In some embodiments, targeting and degradation of a receptor tyrosine kinase with a selective depletion complex may be beneficial in treating a variant of cancers. For example, targeting and degrading EGFR with a selective depletion complex comprising an EGFR-binding peptide may be beneficial in treating cancers, such as non-small- cell lung cancer, primary non-small-cell lung cancer, metastatic non-small-cell lung cancer, head and neck cancer, head and neck squamous cell carcinoma, glioblastoma, brain cancer, metastatic brain cancer, colorectal cancer, colon cancer, tyrosine kinase inhibitor (TKI)-resistant cancer, cetuximab-resistant cancer, necitumumab-resistant cancer, panitumumab-resistant cancer, local cancer, regionally advanced cancer, recurrent cancer, metastatic cancer, refractory cancer, KRAS wildtype cancer, KRAS mutant cancers, or exon20 mutant non-small-cell lung cancer. [0278] Additionally, the selective depletion complexes of the present disclosure may be well- suited for treatment of CNS-associated disorders such as EGFR-driven brain cancers due to the ability of the selective depletion complexes to penetrate the blood-brain barrier (BBB) and access the CNS via TfR-binding. A selective depletion complex (e.g., comprising a TfR-binding peptide) may facilitate higher BBB penetration.

[0279] In some embodiments, binding and subsequently depleting a target molecule using a selective depletion complex of the present disclosure comprising a target-binding peptide may be used to treat a disease or condition wherein the target molecule is a cell-based or soluble moiety associated with a disease or condition and is expressed or present in diseased tissues or cells. In some embodiments, depletion of the target molecule may be cell type or tissue dependent. For example, depletion of a target molecule may be specific to cells or tissues expressing both the target molecule targeted by the target-binding peptide of the selective depletion complex and the cell surface receptor targeted by the receptor-binding peptide of the selective depletion complex. The degradation and depletion and of the target molecule using a selective depletion complex may prevent, treat, or ameliorate the disease or condition.

[0280] In some embodiments, a target-binding peptide (e.g., a target-binding EGF variant) may comprise a sequence of any one of SEQ ID NO: 317 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494. In some embodiments, a target-binding peptide may comprise a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494, or a fragment thereof. For example, a target-binding peptide may comprise a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494. Examples of target-binding peptides and their corresponding target molecules are provided in TABLE 3.

[0281] In some embodiments, a target-binding peptide (e.g., an EGFR target-binding peptide) may comprise a sequence of any one of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494. In some embodiments, a target-binding peptide (e.g., an EGFR targetbinding peptide) may comprise a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494, or a fragment thereof.

[0282] In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 458 - SEQ ID NO: 494, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of any one of SEQ ID NO: 458 - SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 80% sequence identity with any one of SEQ ID NO: 458 - SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 90% sequence identity with any one of SEQ ID NO: 458 - SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 95% sequence identity with SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence of any one of SEQ ID NO: 458 - SEQ ID NO: 494.

[0283] In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 494, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR targetbinding peptide) comprises a sequence that has at least 80% sequence identity with SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 90% sequence identity with SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence that has at least 95% sequence identity with SEQ ID NO: 494. In some aspects, a target-binding peptide (e.g., an EGFR target-binding peptide) comprises a sequence of SEQ ID NO: 494.

[0284] Examples of EGFR target-binding peptides are provided in TABLE 3.

TABLE 3 - Exemplary Target-Binding Peptides that Bind EGER as a Target

-Ill-

[0285] In some embodiments, a target-binding peptide disclosed herein (e.g., an EGFR targetbinding peptide) comprises a sequence of XlX2X3X4X₅CX6X7X₈X9X10XllX12CX13X14Xl₅GX16CX17YSXl₈X19LX20X21YX22CNCX23X₂4G YX25GX26RCQX27X₂₈X29X3oX3iX32X33 (SEQ ID NO: 314) wherein Xi is any amino acid; X₂ is any amino acid; X3 is any amino acid; X4 is any of Lys, Ser, He, or Vai; X5 is any of Glu, Lys, or Asn; Xe is Asp or Pro; X7 is any amino acid; X₈ is any of Gly, Ser, or Met; X9 is any of His, Leu, or Trp; X10 is Arg or Vai; Xu is any of Gly, Asp, Lys, Arg, Asn, or Met; X12 is Phe or Tyr; X13 is any of Met, Leu, or Phe; X14 is His or Gin; X15 is any of Asp, Arg, Asn; Xi6 is any of Gin, Met, Vai; X17 is any of His, Arg, Asn, Vai; Xi₈ is any of Glu, Lys, Gin, Ser, Ala, Vai; X19 is any of Glu, Ser, or Ala; X20 is Asp or Asn; X21 is any of Lys, Thr, He, or Leu; X22 is He or Trp; X23 is Ser or Thr; X24 is any of Glu, He, or Phe; X25 is any amino acid; X26 is any of Asp, Glu, or Pro; X27 is Ser or Tyr; X2₈ is any of His, Lys, Arg, or Gin; X29 is Asp or Thr; X30 is Leu or Phe; X31 is any of Gly, Asn, Ser, Thr; X32 is any amino acid; and X33 is any of Met, Leu, Phe, Trp.

[0286] An EGFR-binding peptide of the present disclosure may comprise a configuration (e.g., a cysteine configuration), one or more amino acid substitutions, or a combination thereof, relative to an EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317), wherein “relative to” the EGF peptide describes the amino acid positions of the EGFR-binding peptide relative to the amino acid positions of the EGF peptide. For example, an EGFR-binding peptide of the present disclosure may comprise 53 amino acids described by positions 1-53 relative to positions 1-53 of SEQ ID NO: 317. In another example, an EGFR-binding peptide of the present disclosure may comprise 50 amino acids described by positions 1-50 relative to positions 1-50 of SEQ ID NO: 317. In some embodiments, an EGFR-binding peptide may comprise additional amino acid positions on the N terminus of the peptide relative to an EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317) which can be described as amino acid positions 0 through -n relative to the EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317). For example, an EGFR-binding peptide may comprise an additional 5 amino acids on the N terminus as compared to an EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317) that can be described as amino acid positions 0, -1, -2, -3, and -4 relative to the EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317). In some embodiments, an EGFR-binding peptide may comprise additional amino acid positions on the C terminus of the peptide which can be described as amino acid positions of the last position number +1 through the last position number +n (e.g., 54 through 53+n relative to SEQ ID NO: 317) relative to the EGF peptide (e.g., a human EGF peptide of SEQ ID NO: 317). For example, an EGFR-binding peptide may comprise an additional 5 amino acids on the C terminus as compared to an EGF peptide of SEQ ID NO: 317 that can be described as amino acid positions 54, 55, 56, 57, and 58 relative to the EGF peptide of SEQ ID NO: 317. The amino acid positions of an EGFR-binding peptide relative to an EGF variant may describe a position of a specific amino acid (e.g., a position of a cysteine amino acid). The amino acid positions of an EGFR-binding peptide relative to an EGF variant may describe a position of an amino acid substitution using amino acid substitution nomenclature of “(the original amino acid)(the position of the amino acid substitution)(the substituted amino acid).” For example, an EGFR-binding peptide with a DI 1R amino acid substitution relative to a human EGF peptide of SEQ ID NO: 317 describes the amino acid substitution of R in place of D at position 11 of the EGFR-binding peptide.

[0287] In some embodiments, an EGFR target-binding peptide may comprise a cysteine configuration of that of an EGF peptide (e.g., a human EGF peptide). For example, an EGFR target-binding peptide may comprise cysteine amino acids at positions 6, 14, 20, 31, 33, and 42 of the EGFR target-binding peptide, not counting tags, linkers, or other components complexed with the EGFR target-binding peptide. In some embodiments, an EGFR target-binding peptide may comprise cysteine residues spaced by 7 amino acid residues between a first cysteine and a second cysteine, 5 amino acid residues between the second cysteine and a third cysteine, 10 amino acid residues between the third cysteine and a fourth cysteine, 1 amino acid residue between the fourth cysteine and a fifth cysteine, and 8 amino acid residues between the fifth cysteine and a sixth cysteine (SEQ ID NO: 711). An EGFR target-binding peptide may comprise a truncation relative to an EGF peptide. In some embodiments, an EGFR target-binding peptide may comprise 1, 2, 3, 4, or 5 amino acids truncated from the C-terminus relative to EGF. In some embodiments, an EGFR target-binding peptide may comprise 1, 2, 3, 4, or 5 amino acids truncated from the N-terminus relative to EGF. An EGFR target-binding peptide may comprise at least 70%, at least 75%, at least 77%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, or at least 96% sequence identity to an EGF peptide (e.g., a human EGF) and may further comprise at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the EGF peptide. The EGFR target-binding peptide may comprise amino acid substitutions DI 1R, I23S, V35E, S51P, L52E, R53E, M21R, A30W, I38D, W49R, V34S, Q43I, Q43V, Q43W, Q43Y, K48N, K48T, K48A, K48L, E51S, E51H, L52H, R53H, or combinations thereof, relative to human EGF. For example, an EGFR target-binding peptide may comprise substitutions V34S, K48T, E51S, or any combination thereof, relative to human EGF. In another example, an EGFR target-binding peptide may comprise substitutions M21R, A30W, I38D, W49R, or combinations thereof, relative to human EGF. In another example, an EGFR target-binding peptide may comprise substitutions DI 1R, I23S, V35E, S51P, L52E, R53E, or combinations thereof, relative to human EGF. In another example, an EGFR target-binding peptide may comprise substitutions E51H, L52H, R53H, or combinations thereof, relative to human EGF.

Cystine-Dense Peptides

[0288] In some embodiments, TfR-binding peptides or PD-Ll-binding peptides or targetbinding peptides (e.g., EGFR target-binding peptides) of the present disclosure comprise one or more Cys, or one or more disulfide bonds. In some embodiments, the TfR-binding peptides or the target-binding peptides (e.g., EGFR target-binding peptides) are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins. As used herein, the term “peptide” is considered to be interchangeable with the terms “knotted peptide”, “cystine-dense peptide”, “CDP”, and “hitchin”. (See e.g., Correnti et al. Screening, large-scale production, and structure -based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278). [0289] The TfR-binding peptides of the present disclosure, or derivatives, fragments, or variants thereof, can have an affinity and selectively for TfR, or a derivative or analog thereof. The target-binding peptides of the present disclosure, or derivatives, fragments, or variants thereof, can have an affinity and selectively for a target molecule. In some cases, the TfR-binding peptides of the present disclosure can be engineered using site-saturation mutagenesis (SSM) to exhibit improved TfR-binding properties or promote transcytosis or endocytosis more effectively. In some cases, the target-binding peptides of the present disclosure can be engineered using site-saturation mutagenesis (SSM) to exhibit improved target-binding properties. In some cases, the peptides of the present disclosure are cystine-dense peptides (CDPs), related to knotted peptides or hitchin-derived peptides or knottin-derived peptides. The TfR-binding peptides can be cystine-dense peptides (CDPs). Hitchins can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity [1-4], 2-5, 3-6 indicating that the first cysteine residue forms a disulfide bond with the fourth residue, the second with the fifth, and the third cysteine residue with the sixth. The brackets in this nomenclature indicate cysteine residues form the knotting disulfide bond. (See e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278). Knottins can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity 1-4, 2-5, [3-6]. Knottins are a class of peptides, usually ranging from about 20 to about 80 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and can contain beta strands and other secondary structures. The presence of the disulfide bonds gives knottins and hitchins remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. In some cases, the peptides described herein can be derived from knotted peptides. The amino acid sequences of peptides as disclosed herein can comprise a plurality of cysteine residues. In some cases, at least cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. In some cases, all cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. As described herein, the term “knotted peptide” can be used interchangeably with the terms “cystine-dense peptide”, “CDP”, or “peptide”.

[0290] Provide herein are methods of identification, maturation, characterization, and utilization of CDPs that bind the transferrin receptor and allow selection, optimization and characterization of CDP-TfR binding peptides that can be used in selective depletion complexes, including for use as bioactive molecules at therapeutically relevant concentrations in a subject (e.g., a human or non-human animal). This disclosure demonstrates the utility of CDPs as a diverse scaffold family that can be screened for applicability to modem drug discovery strategies. CDPs comprise alternatives to existing biologies, primarily antibodies, which can bypass some of the liabilities of the immunoglobulin scaffold, including poor tissue permeability, immunogenicity, and long serum half-life that can become problematic if toxicities arise. Peptides of the present disclosure in the 20-80 amino acid range represent medically relevant therapeutics that are midsized, with many of the favorable binding specificity and affinity characteristics of antibodies but with improved stability, reduced immunogenicity, and simpler manufacturing methods. The intramolecular disulfide architecture of CDPs provides particularly high stability metrics, reducing fragmentation and immunogenicity, while their smaller size could improve tissue penetration or cell penetration and facilitate tunable serum half-life. Disclosed herein are peptides representing candidate peptides that can serve as vehicles for delivering target molecules to endocytic compartments.

[0291] In some embodiments, TfR-binding peptides can be engineered peptides. An engineered peptide can be a peptide that is non-naturally occurring, artificial, isolated, synthetic, designed, or recombinantly expressed. In some embodiments, the TfR-binding peptides of the present disclosure comprise one or more properties of CDPs, knotted peptides, or hitchins, such as stability, resistance to proteolysis, resistance to reducing conditions, and/or ability to cross the blood brain barrier. In some embodiments, the target-binding peptides of the present disclosure comprise one or more properties of CDPs, knotted peptides, or hitchins, such as stability, resistance to proteolysis, or resistance to reducing conditions.

[0292] CDPs can be advantageous for delivery to the CNS, as compared to other molecules such as antibodies due to smaller size, greater tissue or cell penetration, lack of Fc function, and quicker clearance from serum, and as compared to smaller peptides due to resistance to proteases (both for stability and for immunogenicity reduction). In some embodiments, the TfR-binding peptides or target-binding peptides of the present disclosure (e.g., CDPs, knotted peptides, or hitchins), selective depletion complexes (e.g., comprising one or more TfR-binding peptides and one or more target-binding peptides), or engineered TfR-binding fusion peptides (e.g., comprising one or more TfR-binding peptides and one or more peptides) can have properties that are superior to TfR-binding antibodies or target-binding antibodies. For example, the peptides and complexes described herein can provide superior, deeper, and/or faster tissue or cell penetration to cells and targeted tissues (e.g., brain parenchyma penetration, solid tumor penetration) and faster clearance from non- targeted tissues and serum. The TfR-binding peptides, target-binding peptides, selective depletion complexes, or TfR-binding fusion peptides of this disclosure can have lower molecular weights than TfR-binding antibodies or targetbinding antibodies. The lower molecular weight can confer advantageous properties on the TfR- binding peptides, target-binding peptides, selective depletion complexes, or TfR-binding fusion peptides of this disclosure as compared to TfR-binding antibodies or target-binding antibodies. For example, the TfR-binding peptides, selective depletion complexes, or TfR-binding fusion peptides of this disclosure can penetrate a cell or tissue more readily than an anti-TfR antibody or can have lower molar dose toxicity than an anti-TfR antibody. The TfR-binding peptides, target-binding peptides, selective depletion complexes, or TfR-binding fusion peptides of this disclosure can be advantageous for lacking the Fc function of an antibody. The TfR-binding peptides, target-binding peptides, selective depletion complexes, or TfR-binding fusion peptides of this disclosure can be advantageous for allowing higher concentrations, on a molar basis, of formulations.

[0293] In some embodiments, CDPs or knotted peptides, including engineered, non-naturally occurring CDPs and those found in nature (e.g., a target-binding peptide), can be conjugated to, linked to, or fused to the TfR-binding peptides of the present disclosure, such as those described in TABLE 1, to selectively deliver a target molecule to an endocytic compartment of cell. The cell can be a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, bone marrow cell, or any combination thereof. The cell can be any cell that expresses TfR. An engineered peptide can be a peptide that is non-naturally occurring, artificial, synthetic, designed, or recomb inantly expressed. In some embodiments, a TfR-binding peptide of the present disclosure, or a complex comprising a TfR-binding peptide (e.g., a selective depletion complex), enables TfR-mediated transcytosis and/or cellular endocytosis, and the additional CDP or knotted peptide that is conjugated to, linked to, or fused to TfR-binding peptide can selectively target a molecule (e.g., an enzyme or other protein of interest) in a cell associated with a disease or condition. In some cases, the cell is a cancer cell. Cancers can include breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, nonHodgkin lymphoma, myeloma, blood-cell-derived cancer, spleen cancer, cancers of the salivary gland, kidney cancer, muscle cancers, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, sarcoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, bone marrow cell cancers, or skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, a neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, and diffuse intrinsic pontine glioma (DIPG), or a CMYC-overexpressing cancer. In some cases, other CDP or knotted peptides (e.g., those found in nature) are conjugated to, linked to, or fused to TfR- binding peptides and are capable of localizing TfR-binding peptides across the blood brain barrier to deliver TfR-binding peptides to target cells in the central nervous system.

[0294] CDPs (e.g., knotted peptides or hitchins) are a class of peptides, usually ranging from about 11 to about 81 amino acids in length that are often folded into a compact structure. Knotted peptides are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and can contain beta strands, alpha helices, and other secondary structures. The presence of the disulfide bonds gives knotted peptides remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. The presence of a disulfide knot can provide resistance to reduction by reducing agents. The rigidity of knotted peptides also allows them to bind to targets without paying the “entropic penalty” that a floppy peptide accrues upon binding a target. For example, binding is adversely affected by the loss of entropy that occurs when a peptide binds a target to form a complex. Therefore, “entropic penalty” is the adverse effect on binding, and the greater the entropic loss that occurs upon this binding, the greater the “entropic penalty.” Furthermore, unbound molecules that are flexible lose more entropy when forming a complex than molecules that are rigidly structured, because of the loss of flexibility when bound up in a complex. However, rigidity in the unbound molecule also generally increases specificity by limiting the number of complexes that molecule can form. The peptides can bind targets with antibody-like affinity, or with nanomolar or picomolar affinity. A wider examination of the sequence structure and sequence identity or homology of knotted peptides reveals that they have arisen by convergent evolution in all kinds of animals and plants. In animals, they are often found in venoms, for example, the venoms of spiders and scorpions and have been implicated in the modulation of ion channels. The knotted proteins of plants can inhibit the proteolytic enzymes of animals or have antimicrobial activity, suggesting that knotted peptides can function in molecular defense systems found in plants.

[0295] A peptide of the present disclosure (e.g., a target-binding peptide, a TfR-binding peptide, or a selective depletion complex) can comprise a cysteine amino acid residue. In some embodiments, the peptide has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine amino acid residues. In some embodiments, the peptide has at least 6 cysteine amino acid residues. In some embodiments, the peptide has at least 8 cysteine amino acid residues. In other embodiments, the peptide has at least 10 cysteine amino acid residues, at least 12 cysteine amino acid residues, at least 14 cysteine amino acid residues or at least 16 cysteine amino acid residues.

[0296] A knotted peptide can comprise disulfide bridges. A knotted peptide can be a peptide wherein 5% or more of the residues are cysteines forming intramolecular disulfide bonds. A disulfide-linked peptide can be a drug scaffold. In some embodiments, the disulfide bridges form a knot. A disulfide bridge can be formed between cysteine residues, for example, between cysteines 1 and 4, 2 and 5, or, 3 and 6. In some embodiments, one disulfide bridge passes through a loop formed by the other two disulfide bridges, for example, to form the knot. In other embodiments, the disulfide bridges can be formed between any two cysteine residues. [0297] The present disclosure further includes peptide scaffolds that, e.g., can be used as a starting point for generating additional peptides. In some embodiments, these scaffolds can be derived from a variety of knotted peptides (such as CDPs or knotted peptides or hitchins). In certain embodiments, CDPs (e.g., knotted peptides or hitchins) are assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks, and optionally contain beta strands and other secondary structures such as an alpha helix. For example, CDPs (e.g., knotted peptides) include, in some embodiments, small disulfide-rich proteins characterized by a disulfide through disulfide knot. This knot can be, e.g., obtained when one disulfide bridge crosses the macrocycle formed by two other disulfides and the interconnecting backbone. In some embodiments, the knotted peptides can include growth factor cysteine knots or inhibitor cysteine knots. Other possible peptide structures include peptide having two parallel helices linked by two disulfide bridges without P-sheets (e.g., hefutoxin). [0298] Some peptides of the present disclosure can comprise at least one amino acid residue in an L configuration. A peptide can comprise at least one amino acid residue in D configuration. In some embodiments, a peptide is 15-75 amino acid residues long. In other embodiments, a peptide is 11-55 amino acid residues long. In still other embodiments, a peptide is 11-65 amino acid residues long. In further embodiments, a peptide is at least 20 amino acid residues long. [0299] Some CDPs (e.g., knotted peptides) can be derived or isolated from a class of proteins known to be present or associated with toxins or venoms. In some cases, the peptide can be derived from toxins or venoms associated with scorpions or spiders. The peptide can be derived from venoms and toxins of spiders and scorpions of various genus and species. For example, the peptide can be derived from a venom or toxin of the Leiurus quinquestriatus hebraeus, Buthus occitanus tunetanus, Hottentotta judaicus, Mesobuthus eupeus, Buthus occitanus israelis, Hadrurus gertschi, Androctonus australis, Centruroides noxius, Heterometrus laoticus, Opistophthalmus carinatus, Haplopelma schmidti, Isometrus maculatus, Haplopelma huwenum, Haplopelma hainanum, Haplopelma schmidti, Agelenopsis aperta, Haydronyche versuta, Selenocosmia huwena, Heteropoda venatoria, Grammostola rosea, Ornithoctonus huwena, Hadronyche versuta, Atrax robustus, Angelenopsis aperta, Psalmopoeus cambridgei, Hadronyche infensa, Paracoelotes luctosus, and Chilobrachys jingzhaoor another suitable genus or species of scorpion or spider. In some cases, a peptide can be derived from a Buthus martensii Karsh (scorpion) toxin.

[0300] In some embodiments, a peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-L1 -binding peptide, a target-binding peptide, or a selective depletion complex) can comprise a sequence having cysteine residues at one or more of corresponding positions 11, 12, 13, 14, 19, 20, 21, 22, 36, 38, 39, 41, for example with reference to SEQ ID NO: 96. In some embodiments, a peptide comprises Cys at corresponding positions 11, 12, 19, 20, 36, 39, or any combination thereof. For example, in certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 11. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 12. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 13. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 14. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 19. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 21. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 22. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 36. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 38. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 39. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 41. In some embodiments, the first cysteine residue in the sequence can be disulfide bonded with the 4th cysteine residue in the sequence, the 2nd cysteine residue in the sequence can be disulfide bonded to the 5th cysteine residue in the sequence, and the 3rd cysteine residue in the sequence can be disulfide bonded to the 6th cysteine residue in the sequence. Optionally, a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system. In some embodiments, the peptides disclosed herein can have one or more cysteines mutated to serine.

[0301] In some embodiments, peptides of the present disclosure (e.g., TfR-binding peptides, target-binding peptides, or selective depletion complexes) comprise at least one cysteine residue. In some embodiments, peptides of the present disclosure comprise at least two cysteine residues. In some embodiments, peptides of the present disclosure comprise at least three cysteine residues. In some embodiments, peptides of the present disclosure comprise at least four cysteine residues. In some embodiments, peptides of the present disclosure comprise at least five cysteine residues. In some embodiments, peptides of the present disclosure comprise at least six cysteine residues. In some embodiments, peptides of the present disclosure comprise at least ten cysteine residues. In some embodiments, a peptide of the present disclosure comprises six cysteine residues. In some embodiments, a peptide of the present disclosure comprises seven cysteine residues. In some embodiments, a peptide of the present disclosure comprises eight cysteine residues.

[0302] In some embodiments, a peptide of the present disclosure (e.g., a TfR-binding peptide, a target-binding peptide, or a selective depletion complex) comprises an amino acid sequence having cysteine residues at one or more positions, for example with reference to SEQ ID NO: 96. In some embodiments, the one or more cysteine residues are located at any one of the corresponding amino acid positions 6, 10, 20, 34, 44, 48, or any combination thereof. In some aspects of the present disclosure, the one or more cysteine (C) residues participate in disulfide bonds with various pairing patterns (e.g., C10-C20). In some embodiments, the corresponding pairing patterns are C6-C48, C10-C44, and C20-C34. In some embodiments, the peptides as described herein comprise at least one, at least two, or at least three disulfide bonds. In some embodiments, at least one, at least two, or at least three disulfide bonds are arranged according to the corresponding C6-C48, C10-C44, and C20-C34 pairing patterns, or a combination thereof. In some embodiments, peptides as described herein comprise three disulfide bonds with the corresponding pairing patterns C6-C48, C10-C44, and C20-C34.

[0303] In certain embodiments, a peptide (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a target-binding peptide, or a selective depletion complex) comprises a sequence having a cysteine residue at corresponding position 6. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 10. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 34. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 44. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 50. In some embodiments, the first cysteine residue in the sequence is disulfide bonded with the last cysteine residue in the sequence. In some embodiments, the second cysteine residue in the sequence is disulfide bonded with the second to the last cysteine residue in the sequence. In some embodiments, the third cysteine residue in the sequence is disulfide bonded with the third to the last cysteine residue in the sequence and so forth. [0304] In some embodiments, the first cysteine residue in the sequence is disulfide bonded with the 6th cysteine residue in the sequence, the 2nd cysteine residue in the sequence is disulfide bonded to the 5th cysteine residue in the sequence, and the 3rd cysteine residue in the sequence is disulfide bonded to the 4th cysteine residue in the sequence. Optionally, a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system. In some embodiments, the peptides disclosed herein have one or more cysteines mutated to serine.

[0305] In some embodiments, a peptide (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a target-binding peptide, or a selective depletion complex) comprises no cysteine or disulfides. In some embodiments, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more cysteine or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with serine residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with threonine residues.

[0306] In some embodiments, a peptide (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a target-binding peptide, or a selective depletion complex) comprises no Cys or disulfides. In some embodiments, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues.

[0307] In some instances, one or more or all of the methionine residues in the peptide are replaced by leucine or isoleucine. In some instances, one or more or all of the tryptophan residues in the peptide are replaced by phenylalanine or tyrosine. In some instances, one or more or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, the N-terminus of the peptide is blocked, such as by an acetyl group. Alternatively or in combination, in some instances, the C-terminus of the peptide is blocked, such as by an amide group. In some embodiments, the peptide is modified by methylation on free amines. [0308] For example, full methylation can be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.

[0309] In some embodiments, the peptides or peptide complexes as described herein target and/or penetrate a TfR-expressing cellular layer or barrier and/or the membrane of a TfR- expressing cell. In some embodiments, a peptide targets and/or penetrates a cell membrane of a cell, wherein said cell is located in the CNS such as the brain. For example, a peptide complex comprising a TfR-binding peptide and one or more active agents (e.g., a therapeutic or diagnostic compound) crosses a cellular barrier (e.g., BBB) via vesicular transcytosis, and subsequently targets and/or penetrates the cell membrane of a cell located within the CNS to deliver said one or more active agents to that cell.

[0310] In various embodiments, a selective depletion complex comprising a TfR-binding peptide and a target-binding peptide binds a TfR-expressing cell located in the gastrointestinal tract, spleen, liver, kidney, muscle, bone marrow, brain, or skin. In some cases, the TfR- expressing cell is a tumor cell, an immune cell, an erythrocyte, an erythrocyte precursor cell, a stem cell, a bone marrow cell, or stem cell. In some cases, the TfR-binding peptide is responsible for targeting the cell, e.g., in cases where the cell is overexpressing a TfR. In various embodiments, a peptide complex as described herein comprising a TfR-binding peptide conjugated to, linked to, or fused to a target-binding peptide binds a cell located within various organs such as the spleen, brain, liver, kidney, muscle, bone marrow, gastrointestinal tract, or skin.

[0311] In some cases, the target-binding peptides promotes endocytosis of a target molecule. In some aspects, a peptide or peptide complex (e.g., peptide conjugate or fusion peptide) of the present disclosure is used to target a target molecule in order to exert a certain biological (e.g., therapeutic) effect. In some aspects, a selective depletion complex (e.g., a complex comprising a TfR-binding peptide and a target-binding peptide) of the present disclosure is used to promote endocytosis of a target molecule into said cell to exert a certain biological effect (e.g., selective depletion of the target molecule).

Peptide Linkers

[0312] The peptides of the presented disclosure (e.g., TfR-binding peptides, PD-L1 binding peptides, target-binding peptides such as EGFR target-binding peptides, selective depletion complexes, or combinations thereof) can be dimerized in numerous ways. For example, a TfR- binding peptide can be dimerized with a target-binding peptide via a peptide linker to form a selective depletion complex. In some embodiments, a peptide linker does not disturb the independent folding of peptide domains (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, or an EGFR target-binding peptide). In some embodiments, a peptide linker can comprise sufficient length to the peptide complex so as to facilitate contact between a target molecule and a TfR via the peptide complex (e.g., a selective depletion complex). In some embodiments, a peptide linker does not negatively impact manufacturability (synthetic or recombinant) of the peptide complex (e.g., the selective depletion complex). In some embodiments, a peptide linker does not impair post-synthesis chemical alteration (e.g., conjugation of a fluorophore or albumin-binding chemical group) of the peptide complex (e.g., the selective depletion complex). In some embodiments, cellular receptor-binding peptide is conjugated to the target-binding peptide via a polymer linker. In some embodiments, the polymer linker is a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer comprising proline, alanine, serine, or a combination thereof, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, a palmitic acid, an albumin, or an albumin binding molecule.

[0313] In some embodiments, a peptide linker can connect the C-terminus of a first peptide (e.g., an EGFR target-binding peptide, a TfR-binding peptide, or a half-life modifying peptide) to the N-terminus of a second peptide (e.g., an EGFR target-binding peptide, a TfR-binding peptide, or a half-life modifying peptide). In some embodiments, a peptide linker can connect the C-terminus of the second peptide (e.g., an EGFR target-binding peptide, a TfR-binding peptide, or a half-life modifying peptide) to the N-terminus of a third peptide (e.g., an EGFR target-binding peptide, a TfR-binding peptide, or a half-life modifying peptide). For example, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to the N-terminus of a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to form a selective depletion complex. In another example, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a TfR- binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to the N-terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to form a selective depletion complex. In another example, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a TfR- binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to the N-terminus of a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192 SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710) and the C- terminus of the half-life extending peptide to the N-terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to form a selective depletion complex. In another example, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to the N-terminus of a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192, SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710) and a second linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C- terminus of the half-life extending peptide to the N-terminus of a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to form a selective depletion complex. In another example, a first linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to the N-terminus of a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192, SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710) and a second linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of the half-life extending peptide to the N-terminus of a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to form a selective depletion complex. In another example, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192, SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710) to the N- terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) and a second linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of the targetbinding peptide to the N-terminus of a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) to form a selective depletion complex. In another embodiment, a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192, SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710) to the N-terminus of a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) and a second linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) can connect the C-terminus of the TfR-binding peptide to the N- terminus of a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) to form a selective depletion complex.

[0314] In some embodiments, a linker can comprise a Tau-theraphotoxin-Hsla, also known as DkTx (double-knot toxin), extracted from a native knottin-knottin dimer from Haplopelma schmidti (e.g., SEQ ID NO: 139). The linker can lack structural features that would interfere with dimerizing independently functional CDPs (e.g., a TfR-binding CDP and an EGFR target- binding CDP). In some embodiments, a linker can comprise a glycine-serine (Gly-Ser or GS) linker (e.g., SEQ ID NO: 129 - SEQ ID NO: 138 or SEQ ID NO: 141 or SEQ ID NO: 195 - SEQ ID NO: 218 or SEQ ID NO: 538). Gly-Ser linkers can have minimal chemical reactivity and can impart flexibility to the linker. Serines can increase the solubility of the linker or the peptide complex, as the hydroxyl on the side chain is hydrophilic. In some embodiments, a linker can be derived from a peptide that separates the Fc from the Fv domains in a heavy chain of human immunoglobulin G (e.g., SEQ ID NO: 140). In some embodiments, a linker derived from a peptide from the heavy chain of human IgG can comprise a cysteine to serine mutation relative to the native IgG peptide.

[0315] In some embodiments, peptides of the present disclosure can be dimerized using an immunoglobulin heavy chain Fc domain. In some embodiments, the half-life of peptides of the present disclosure can be extended using an Fc domain, such Fc domains are described herein and also referred to as a “half-life extender”. In some embodiments, an Fc domain can serve as a linker. These Fc domains can be used to dimerize functional domains (e.g., a TfR-binding peptide and a target-binding peptide, or a PD-L1 -binding peptide and a target-binding peptide), either based on antibodies or other otherwise soluble functional domains. In some embodiments, dimerization can be homodimeric if the Fc sequences are native. In some embodiments, dimerization can be heterodimeric by mutating the Fc domain to generate a “knob-in-hole” format where one Fc CH3 domain contains novel residues (knob) designed to fit into a cavity (hole) on the other Fc CH3 domain. A first peptide domain (e.g., a TfR-binding peptide, a PD- L1 -binding peptide, or an EGFR target-binding peptide) can be coupled to the knob, and a second peptide domain (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, or an EGFR target-binding peptide) can be coupled to the hole. Knob+knob dimers can be highly energetically unfavorable. A purification tag can be added to the “knob” side to remove hole+hole dimers and select for knob+hole dimers. In some embodiments, dimerization can be heterodimeric by mutating the Fc domain to generate an “electrostatic steering” effect wherein one Fc CH3 domain (“Chain 1”) contains mutations that manipulate the distribution of charges at the interface, supporting electrostatic interactions with a paired Fc CH3 domain (“Chain 2”) containing mutations creating a reciprocal charge distribution to that of Chain 1. A first peptide domain (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, or an EGFR target-binding peptide) can be coupled to Chain 1 of an engineered pair, and a second peptide domain (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, or an EGFR target-binding peptide) can be coupled to Chain 2 of an electrostatic pair. Homodimers of Chain 1 or Chain 2 can be highly energetically unfavorable, promoting selective secretion of heterodimers.

[0316] The peptide peptides of the present disclosure (e.g., the target-binding peptides such as EGFR target-binding, TfR-binding peptides, or selective depletion complexes) can be linked to another peptide (e.g., a target-binding peptide such as an EGFR target-binding peptide, a TfR- binding peptide, a selective depletion complex, or a half-life modifying peptide) at the N- terminus or C-terminus. In some embodiments, one or more peptides can be linked or fused via a peptide linker (e.g., a peptide linker comprising a sequence of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541). For example, a TfR-binding peptide can be fused to a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) via a peptide linker of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541. A peptide linker (e.g., a linker connecting a TfR-binding peptide, a target-binding peptide, a half-life modifying peptide, or combinations thereof) can have a length of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues. A peptide linker (e.g., a linker connecting a TfR-binding peptide, a target-binding peptide such as an EGFR target-binding peptide, a half-life modifying peptide, or combinations thereof) can have a length of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 3 to about 5, from about 3 to about 10, from about 3 to about 15, from about 3 to about 20, from about 3 to about 25, from about 5 to about 10, from about 5 to about 15, from about 5 to about 20, from about 5 to about 25, from about 10 to about 15, from about 10 to about 20, from about 10 to about 25, from about 15 to about 20, from about 15 to about 25, from about 20 to about 25, from about 20 to about 30, from about 20 to about 35, from about 20 to about 40, from about 20 to about 45, from about 20 to about 50, from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 10 to about 40, from about 10 to about 30, from about 50 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, or from about 500 to about 600 amino acid residues.

[0317] In some embodiments, a first peptide (e.g., a TfR-binding peptide or a PD-Ll-binding peptide) and a second peptide (e.g., an EGFR target-binding peptide) can be connected by a flexible peptide linker. A flexible linker can provide rotational freedom between the first peptide and the second peptide and can allow the first peptide and the second peptide to bind their respective targets (e.g., a transferrin receptor and an EGFR target molecule) with minimal strain. In some embodiments, a peptide linker can have a persistence length of no more than 6 A, no more than 7 A, no more than 8 A, no more than 9 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, or no more than 50 A. In some embodiments, a peptide linker can have a persistence length of no more than 6 A, no more than 7 A, no more than 8 A, no more than 9 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, no more than 50 A, no more than 75 A, no more than 100 A, no more than 150 A, no more than 200 A, no more than 250 A, or no more than 300 A. In some embodiments, a peptide linker can have a persistence length of from about 4 A to about 100 A, from about 4 A to about 50 A, from about 4 A to about 20 A, from about 4 A to about 10 A, from about 10 A to about 20 A, from about 20 A to about 30 A, from about 30 A to about 50 A, or from about 50 A to about 100 A. The persistence length of the linker can be a measure of the flexibility of the peptide linker and can be quantified as the peptide length over which correlations in the direction of the tangent are lost.

[0318] In some embodiments, the peptide linker is derived from an immunoglobulin peptide. In some embodiments, the peptide linker is derived from a double-knot toxin peptide.

[0319] In some embodiments, a peptide linker can be selected based on a desired linker length, hydrodynamic radius, chromatographic mobility, posttranslational modification propensity, or combinations thereof. In some embodiments, a linker separating two or more functional domains of a peptide complex (e.g., separating a TfR-binding peptide and an EGFR target-binding peptide) can comprise a large, stable, globular domain, for example to reduce a propensity for glomerular filtration. In some embodiments, a linker separating two or more functional domains of a peptide complex (e.g., separating a TfR-binding peptide and an EGFR target-binding peptide) can comprise a small, flexible linker, for example to reduce the hydrodynamic radius of the complex for use in tight spaces like dense-core tumor stroma. Examples of selective depletion complexes formed from a single polypeptide chain comprising a target-binding peptide and a receptor-binding peptide connected via a peptide linker are illustrated in FIG. 15 and FIG. 16. In some embodiments, a peptide linker can support independent folding of the two or more functional domains and may not inhibit interactions between the two or more functional domains and their binding targets (e.g., between a TfR-binding peptide and TfR or between a target-binding peptide and a target molecule).

[0320] In some embodiments, a peptide can be appended to the N-terminus of any peptide of the present disclosure following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 129) spacer. In some embodiments, a peptide (e.g., an EGFR target-binding peptide) can be appended to either the N-terminus or C-terminus of any peptide disclosed herein (e.g., a TfR-binding peptide or a PD-L1 -binding peptide) using a peptide linker such as G_xS_y (SEQ ID NO: 130) peptide linker, wherein x and y can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 131), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 132), GGGGG (SEQ ID NO: 133), GSGSGSGS (SEQ ID NO: 134), GSGG (SEQ ID NO: 135), GGGGS (SEQ ID NO: 136), GGGS (SEQ ID NO: 129), GGS (SEQ ID NO: 137), GGGSGGGSGGGS (SEQ ID NO: 138), or a variant or fragment thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 139) from DkTx, and EPKSSDKTHT (SEQ ID NO: 140) from human IgG3 can be used as a peptide linker. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 141). In some embodiments, the peptide linker comprises a linker of any one of SEQ ID NO: 195 - SEQ ID NO: 218. Examples of peptide linkers compatible with the target depletion complexes of the present disclosure are provided in TABLE 4. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.

[0321] In some embodiments, a tag peptide (e.g., a peptide of any one of SEQ ID NO: 142 - SEQ ID NO: 147) can be appended to the peptide (e.g., a target-binding peptide, a TfR-binding peptide, or a selective depletion complex) at any amino acid residue. In further embodiments, the tag peptide (e.g., a peptide of any one of SEQ ID NO: 142 - SEQ ID NO: 147) can be appended to the peptide at any amino acid residue without interfering with TfR-binding activity, target-binding activity, selective depletion activity, or a combination thereof. In some embodiments, the tag peptide is appended via conjugation, linking, or fusion techniques. In other embodiments, a peptide (e.g., an EGFR target-binding peptide) can be appended to a second peptide (e.g., a TfR-binding peptide or a PD-L1 -binding peptide) at any amino acid residue. In further embodiments, the peptide (e.g., an EGFR target-binding peptide) can be appended to the second peptide (e.g., a TfR-binding peptide or a PD-Ll-binding peptide) at any amino acid residue without interfering with TfR-b inding activity, target-binding activity, selective depletion activity, or a combination thereof. In some embodiments, the peptide is appended via conjugation, linking, or fusion techniques. In other embodiments, the peptide (e.g., an EGFR target-binding peptide) can be appended to the second peptide (e.g., a TfR-binding peptide or a PD-L1 -binding peptide) at any amino acid residue.

TABLE 4 - Exemplary Peptide Linkers

[0322] A peptide complex (e.g., a selective depletion complex (SDC)) may comprise multiple polypeptide chains. In some embodiments, a selective depletion complex may comprise two or more polypeptide chains. For example, a target-binding peptide and a receptor-binding peptide may be complexed via a dimerization domain to form a selective depletion complex. In some embodiments, the dimerization domain comprises an Fc domain. The dimerization domain may be a heterodimerization domain or a homodimerization domain. Examples of selective depletion complexes comprising a target-binding peptide and a receptor-binding peptide connected via a dimerization domain (e.g., an Fc homodimerization domain or a knob-in-hole heterodimerization domain) are illustrated in FIG. 15 and FIG. 16.

[0323] A target-binding peptide and a receptor-binding peptide may be complexed by forming a heterodimer via a hetero dimerization domain. The target-binding peptide may be linked or fused to a first heterodimerization domain and the receptor-binding peptide may be linked or fused to a second heterodimerization domain. The first heterodimerization domain may bind to the second heterodimerization domain to form a heterodimeric complex comprising the target-binding peptide and the receptor-binding peptide. For example, the receptor-binding peptide may be linked or fused to an Fc “knob” peptide (e.g., SEQ ID NO: 260, SEQ ID NO: 536, or SEQ ID NO: 707) and the target-binding peptide may be linked or fused to an Fc “hole” peptide (e.g., SEQ ID NO: 261, SEQ ID NO: 537, or SEQ ID NO: 708). In another example, the receptorbinding peptide may be linked or fused to an Fc “hole” peptide (e.g., SEQ ID NO: 261, SEQ ID NO: 537, or SEQ ID NO: 708) and the target-binding may be linked or fused to an Fc “knob” peptide (e.g., SEQ ID NO: 260, SEQ ID NO: 536, or SEQ ID NO: 707). In some embodiments, a receptor-binding peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 222) may form a heterodimer with target-binding peptide via a heterodimerization domain provided in TABLE 5. For example, the receptor-binding peptide may be fused to chain 1 of an Fc pair (e.g., SEQ ID NO: 260) and the target-binding peptide may be fused to chain 2 of the Fc pair (e.g., SEQ ID NO: 261). In another example, the receptor-binding peptide may be fused to chain 2 of an Fc pair (e.g., SEQ ID NO: 263) and the target-binding peptide may be fused to chain 1 of the Fc pair (e.g., SEQ ID NO: 262). A selective depletion complex comprising a heterodimerization domain may form a monovalent selective depletion complex, as shown in FIG. 15 and FIG. 16, or a selective depletion complex comprising a heterodimerization domain may form a multivalent selective depletion complex, as shown in FIG. 15 and FIG. 16. TABLE 5 - Exemplary Heterodimerization Domains

[0324] In some embodiments, a target-binding peptide and a receptor-binding peptide may form a selective depletion complex comprising a homodimer complexed via a homodimerization domain. The target-binding peptide may be linked or fused to the N-terminus of the homodimerization domain and the receptor-binding peptide may be linked or fused to the C- terminus of the homodimerization domain. In some embodiments, the target-binding peptide may be linked or fused to the C-terminus of the homodimerization domain and the receptorbinding peptide may be linked or fused to the N-terminus of the homodimerization domain. In some embodiments, the target-binding peptide and the receptor-binding peptide may both be fused on the N-terminal, or both be fused on the C-terminal end of the homodimerization domain. A selective depletion complex comprising a homodimerization domain may form a multivalent selective depletion complex, as shown in FIG. 15 and FIG. 16. Examples of homodimerization domains that may be used to link or fuse a target-binding peptide to a receptor-binding peptide are provided in TABLE 6.

TABLE 6 - Exemplary Homodimerization Domains

Modification of Peptides

[0325] A peptide can be modified (e.g., chemically modified) one or more of a variety of ways. In some embodiments, the peptide can be mutated to add function, delete function, or modify the in vivo behavior. One or more loops between the disulfide linkages of a peptide (e.g., a TfR- binding peptide, a PD-L1 -binding peptide, a target-binding peptide, or a selective depletion complex) can be modified or replaced to include active elements from other peptides (such as described in Moore and Cochran, Methods in Enzymology, 503, p. 223-251, 2012). In some embodiments, the peptides of the present disclosure (e.g., TfR-binding peptides, target-binding peptides, or selective depletion complexes) can be further functionalized and multimerized by adding an additional functional domain. For example, an albumin-binding domain (ABD) from a Finegoldia magna peptostreptococcal albumin-binding protein (SEQ ID NO: 192, MKLNKKLLMAALAGAIWGGGVNTFAADEPGAIKVDKAPEAPSQELKLTKEEAEKAL KKEKPIAKERLRRLGITSEFILNQIDKATSREGLESLVQTIKQSYLKDHPIKEEKTEETPKY NNLFDKHELGGLGKDKGPGRFDENGWENNEHGYETRENAEKAAVKALGDKEINKSYT ISQGVDGRYYYVLSREEAETPKKPEEKKPEDKRPKMTIDQWLLKNAKEDAIAELKKAGI TSDFYFNAINKAKTVEEVNALKNEILKAHAGKEVNPSTPEVTPSVPQNHYHENDYANIG AGEGTKEDGKKENSKEGIKRKTAREEKPGKEEKPAKEDKKENKKKENTDSPNKKKKE KAALPEAGRRKAEILTLAAASLSSVAGAFISLKKRK). For example, an albumin-binding domain of SEQ ID NO: 193 (LKNAKEDAIAELKKAGITSDFYFNAINKAKTVEEVNALKNEILKA) can be added to a peptide of the present disclosure. In some embodiments, a peptide of the present disclosure can be functionalized with an albumin-binding domain that has been modified for improved albumin affinity, improved stability, reduced immunogenicity, improved manufacturability, or a combination thereof. For example, a peptide can be functionalized with a modified albuminbinding domain of SEQ ID NO: 194 (LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA) or SEQ ID NO: 227 (LKEAKEKAIEELKKAGITSDYYFDLINKAKAVEGVNALKDEILKA) having high thermostability and improved serum half-life compared to the albumin binding domain of SEQ ID NO: 193. In some embodiments, an albumin-binding peptide may be selected based on a desired off rate for albumin. For example, an albumin-binding peptide of SEQ ID NO: 227 may be selected for its faster off rate relative to SEQ ID NO: 194. The albumin-binding domain comprises a simple three-helical structure that would be unlikely to disturb the independent folding of the other peptide domains (e.g., CDP domains). In some embodiments, a functional domain (e.g., an albumin-binding domain) can increase the serum half-life of a peptide or peptide complex of the present disclosure. A functional domain (e.g., an albumin-binding domain) can be included in any orientation relative to the TfR-b inding peptide or the targetbinding peptide. For example, a functional domain can be linked to the TfR-b inding peptide, the target-binding peptide, or in between the TfR-binding peptide and the target-binding peptide. In some embodiments, an albumin binding peptide (e.g., SEQ ID NO: 194 or SEQ ID NO: 227) may be used to link a target-binding peptide to a receptor-binding peptide. An additional functional domain can be linked to one or more peptides (e.g., a TfR-binding peptide, a PD-L1- binding peptide, or a target-binding peptide) via a linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141 or SEQ ID NO: 195 - SEQ ID NO: 218).

[0326] A peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a receptor-binding peptide, a target-binding peptide such as an EGFR target-binding peptide, or a selective depletion complex) may be modified with a signal peptide to mark the peptide for secretion. For example, a peptide may be modified with a signal peptide corresponding to SEQ ID NO: 230 (METDTLLLWVLLLWVPGSTG). In some embodiments, the signal peptide may be appended to an N-terminus or a C-terminus of the peptide. A peptide may be modified for additional stability during translation or secretion. For example, a peptide may be modified with a siderocalin with a furin cleavage site corresponding to SEQ ID NO: 229 (GSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYWGLAGNAILREDKDPQKMYATIY ELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTLGNIKSYPGLTSYLVRWSTNY NQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCI DGGGSRRRRKRSGS). In some embodiments, the siderocalin with the furin cleavage site may be appended to an N-terminus or a C-terminus of the peptide. A peptide may be modified with a signal peptide to mark the peptide for secretion and for additional stability during translation or secretion. For example, a peptide may be modified with a signal peptide and a siderocalin with a furin cleavage site corresponding to SEQ ID NO: 231

(METDTLLLWVLLLWVPGSTGGSQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYWG

LAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGSQPGEFTL GNIKSYPGLTSYLVRWSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFI RFSKSLGLPENHIVFPVPIDQCIDGGGSRRRRKRSGS). In some embodiments, the signal peptide and the siderocalin with the furin cleavage site may be appended to an N-terminus or a C-terminus of the peptide.

[0327] A peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a receptor-binding peptide, a target-binding peptide such as an EGFR target-binding peptide, or a selective depletion complex) may be modified with a signal peptide to mark the peptide for secretion and further have the signal peptide removed without loss of function or binding-properties. For example, a peptide may be modified with a signal peptide corresponding to SEQ ID NO: 230 (METDTLLLWVLLLWVPGSTG) and have the signal peptide removed without loss of function or binding-properties.

[0328] Amino acids of a peptide or a peptide complex (e.g., a TfR-binding peptide, a PD-Ll- binding peptide, a receptor-binding peptide, a target-binding peptide, or a selective depletion complex) can also be mutated, such as to increase half-life, modify, add or delete binding behavior in vivo, add new targeting function, modify surface charge and hydrophobicity, or allow conjugation sites. N-methylation is one example of methylation that can occur in a peptide of the disclosure. In some embodiments, the peptide is modified by methylation on free amines. For example, full methylation can be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.

[0329] The peptides can be modified to add function, such as to graft loops or sequences from other proteins or peptides onto peptides of this disclosure. Likewise, domains, loops, or sequences from this disclosure can be grafted onto other peptides or proteins such as antibodies that have additional function.

[0330] In some embodiments, a selective depletion complex can comprise a tissue targeting domain and can accumulate in the target tissue upon administration to a subject. For example, selective depletion complexes can be conjugated to, linked to, or fused to a molecule (e.g., small molecule, peptide, or protein) with targeting or homing function for a cell of interest or a target protein located on the surface or inside said cell. In some embodiments, selective depletion complexes can be conjugated to, linked to, or fused to a molecule that extends the plasma and/or biological half-life, or modifies the pharmacodynamic (e.g., enhanced binding to a target protein) and/or pharmacokinetic properties (e.g., rate and mode of clearance) of the peptides, or any combination thereof. [0331] A chemical modification can, for instance, extend the half-life of a peptide or change the biodistribution or pharmacokinetic profile. A chemical modification can comprise a polymer, a polyether, polyethylene glycol, a biopolymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain such as palmitate or myristolate, or albumin. A polyamino acid can include, for example, a poly amino acid sequence with repeated single amino acids (e.g., poly glycine), and a poly amino acid sequence with mixed poly amino acid sequences (e.g., Gly-Ala-Gly-Ala (SEQ ID NO: 712)) that can or may not follow a pattern, or any combination of the foregoing.

[0332] The peptides of the present disclosure can be modified such that the modification increases the stability and/or the half-life of the peptides. The attachment of a hydrophobic moiety, such as to the N-terminus, the C-terminus, or an internal amino acid, can be used to extend half-life of a peptide of the present disclosure. The peptides can also be modified to increase or decrease the gut permeability or cellular permeability of the peptide. In some cases, the peptides of the present disclosure show high accumulation in glandular cells of the intestine, demonstrating applicability in the treatment and-or prevention of diseases or conditions of the intestines, such as Crohn’s disease or more generally inflammatory bowel diseases. The peptide of the present disclosure can include post-translational modifications (e.g., methylation and/or amidation and/or glycosylation), which can affect, e.g., serum half-life. In some embodiments, simple carbon chains (e.g., by myristoylation and/or palmitylation) can be conjugated to, linked to, the fusion proteins or peptides. The simple carbon chains can render the fusion proteins or peptides easily separable from the unconjugated material. For example, methods that can be used to separate the fusion proteins or peptides from the unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography. Lipophilic moieties can extend half-life through reversible binding to serum albumin. Conjugated moieties can, e.g., be lipophilic moieties that extend half-life of the peptides through reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative including cholestenes, cholestanes, cholestadienes and oxysterols. In some embodiments, the peptides can be conjugated to, linked to, myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptides of the present disclosure can be coupled (e.g., conjugated, linked, or fused) to a half-life modifying agent. Examples of half-life modifying agents can include, but is not limited to: a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin. In some embodiments, the cellular receptor-binding peptide and the target-binding peptide form a single polypeptide chain. In some embodiments, the peptide complex comprises a dimer dimerized via a dimerization domain. In some embodiments, the distance between the cellular receptor-binding peptide and the target-binding peptide is at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, or at least 100 nm. In some embodiments, the half-life modifying agent can be a serum albumin binding peptide, for example SA21 (SEQ ID NO: 178, RLIEDICLPRWGCLWEDD). In some embodiments, a SA21 peptide can be conjugated or fused to the CDPs of the present disclosure (e.g., any of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64). A SA21 fhsion peptide can include the SA21 TfR-binding peptide complexes disclosed herein (e.g., SEQ ID NO: 181 or SEQ ID NO: 184). The SA21 peptide can comprise a linker sequence for conjugation to, or fusion between, one or more peptides (e.g., SEQ ID NO: 179, GGGGSGGGGSRLIEDICLPRWGCLWEDDGGGGSGGGGS). Exemplary SA21 peptides, fusion peptides, and linkers are provided in TABLE 7. A control SA21 fusion peptide can comprise a control peptide fused to SA21 (e.g., SEQ ID NO: 180 (GSRLIEDICLPRWGCLWEDDGGGGSGGGGSKCLPPGKPCYGATQKIPCCGVCSHNNCT ), SEQ ID NO: 183 (RLIEDICLPRWGCLWEDDGGGGSGGGGSKCLPPGKPCYGATQKIPCCGVCSHNNCT), SEQ ID NO: 182 (GSRLIEDICLPRWGCLWEDDGGGGSGGGGSVRIPVSCKHSGQCLKPCKDAGMRFGKC MNGKCDCTPK), or SEQ ID NO: 185 (RLIEDICLPRWGCLWEDDGGGGSGGGGSVRIPVSCKHSGQCLKPCKDAGMRFGKCMN GKCDCTPK)). Additionally, conjugation of the peptide to a near infrared dye, such as Cy5.5, or to an albumin binder such as Albu-tag can extend serum half-life of any peptide as described herein. In some embodiments, immunogenicity is reduced by using minimal non-human protein sequences to extend serum half-life of the peptide. TABLE 7 - Exemplary TfR-Binding Peptide Complexes Comprising Serum Albumin Binding Peptides

[0333] In some embodiments, the first two N-terminal amino acids (GS) of SEQ ID NO: 1 - SEQ ID NO: 64 serve as a spacer or linker in order to facilitate conjugation or fusion to another molecule, as well as to facilitate cleavage of the peptide from such conjugated to, linked to, or fused molecules. In some embodiments, the fusion proteins or peptides of the present disclosure can be conjugated to, linked to, or fused to other moieties that, e.g., can modify or effect changes to the properties of the peptides.

[0334] In some embodiments, peptides or peptide complexes of the present disclosure can also be conjugated to, linked to, or fused to other affinity handles. Other affinity handles can include genetic fusion affinity handles. Genetic fusion affinity handles can include 6xHis (HHHHHH (SEQ ID NO: 142), 6xHisN (HHHHHHGGGGS (SEQ ID NO: 542)) or GGGGSHHHHHH (SEQ ID NO: 228); immobilized metal affinity column purification possible), FLAG (DYKDDDDK (SEQ ID NO: 143); anti-FLAG immunoprecipitation), “shorty” FLAG (DYKDE (SEQ ID NO: 144); anti-FLAG immunoprecipitation), hemagglutinin (YPYDVPDYA (SEQ ID NO: 145); anti-HA immunoprecipitation), streptavidin binding peptide (e.g., DVEAWLGAR (SEQ ID NO: 146); streptavidin-mediated precipitation), biotinylation tags (e.g., GLNDIFEAQKIEWHE (SEQ ID NO: 543)), or any combination thereof. In some embodiments, peptides of the present disclosure may comprise a biotinylation tag (e.g., SEQ ID NO: 543) and undergo biotinylation by a BirA enzyme. In some embodiments, peptides or peptide complexes of the present disclosure can also be conjugated to, linked to, or fused to an expression tag or sequence to improve protein expression and/or purification. Such expression tags can include genetic fusion expression tags. Genetic fusion expression tags can include siderocalin (SEQ ID NO: 147,

METDTLLLWVLLLWVPGSTGDYKDEHHHHHHGGSQDSTSDLIPAPPLSKVPLQQNFQD NQFQGKWYWGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIR TFVPGSQPGEFTLGNIKSYPGLTSYLVRWSTNYNQHAMVFFKKVSQNREYFKITLYGR TKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDGGGSENLYFQ). [0335] A peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a receptor-binding peptide, a target-binding peptide such as an EGFR target-binding peptide, or a selective depletion complex) may be modified with an affinity handle for to improve protein expression and/or purification and further have the affinity handle removed without loss of function or binding-properties. For example, a peptide may be modified with a 6xHis (HHHHHH (SEQ ID NO: 142) and have the 6xHis (HHHHHH (SEQ ID NO: 142) removed without loss of function or binding-properties. For example, a peptide may be modified with a 6xHisN (HHHHHHGGGGS (SEQ ID NO: 542)) and have the 6xHisN (HHHHHHGGGGS (SEQ ID NO: 542)) removed without loss of function or binding-properties. It is understood that such modification of peptide of the present disclosure can use any length of His affinity handle. Additionally, more than one peptide sequence (e.g., a peptide derived from a toxin or knotted venom protein) can be present on, conjugated to, linked to, or fused with a particular peptide. A peptide can be incorporated into a biomolecule by various techniques. A peptide can be incorporated by a chemical transformation, such as the formation of a covalent bond, such as an amide bond. A peptide can be incorporated, for example, by solid phase or solution phase peptide synthesis. A peptide can be incorporated by preparing a nucleic acid sequence encoding the biomolecule, wherein the nucleic acid sequence includes a subsequence that encodes the peptide. The subsequence can be in addition to the sequence that encodes the biomolecule or can substitute for a subsequence of the sequence that encodes the biomolecule.

Selective Depletion Complexes

[0336] In some embodiments, one or more peptides of the present disclosure can form a selective depletion complex (SDC). In some embodiments, a peptide complex of the present disclosure can be a selective depletion complex (SDC). A selective depletion complex may comprise a target-binding peptide that binds a target molecule and a receptor-binding peptide that binds a cellular receptor (e.g., a cell surface receptor). In some embodiments, the cell surface receptor is a receptor that is endocytosed (e.g., a transferrin receptor or a programmed death-ligand 1). In some embodiments, the cell surface receptor is a receptor that is recycled back to the cell surface following endocytosis. A receptor-binding peptide of the present disclosure may be a transferrin receptor (TfR)-b inding peptide or a programmed death ligand 1 (PD-Ll)-b inding peptide. For example, a selective depletion complex can comprise a TfR- binding peptide and a target-binding peptide. In some embodiments, the receptor-binding peptide (e.g., the TfR-binding peptide or the PD-Ll-binding peptide) and the target-binding peptide can be connected via a linker (e.g., a peptide linker). In some embodiments, the receptor -binding peptide and the target-binding peptide can be directly connected without a linker. In some embodiments, the receptor-binding peptide and the target-binding peptide can be connected via a heterodimerization domain. In some embodiments, the receptor-binding peptide can bind the receptor (e.g., TfR or PD-L1) with high affinity at both extracellular pH (such as about pH 7.4) and at endosomal pH (such as about pH 5.5). In some embodiments, the receptorbinding peptide of a selective depletion complex may be a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64). In some embodiments, the receptor-binding peptide of a selective depletion complex may be a PD-L1 -binding peptide (e.g., any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 141).

[0337] The target-binding peptide can bind a target molecule with an affinity that is pH- dependent. For example, the target-binding molecule can bind to the target molecule with higher affinity at extracellular pH (about pH 7.4) and with lower affinity at a lower endosomal pH (such as about pH 5.5, about pH 5.8, or about pH 6.5). In some embodiments, the target-binding molecule can release the target molecule upon internalization into an endosomal compartment and acidification of the endosome. Such release of the target molecule upon acidification of the endosome can occur at about pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. In some embodiments, release of the target molecule can occur at a pH of from about pH 7.0 to about pH 4.5, from about pH 6.5 to about pH 5.0, or from about pH 6.0 to about pH 5.5. In some embodiments, the receptor-binding peptide binds a receptor (e.g., a receptor that undergoes recycling) with pH-independent binding (e.g., high affinity at extracellular pH and high affinity at endosomal pH) and the target-binding peptide binds the target with pH-dependent binding (e.g., high affinity at extracellular pH and low affinity at endosomal pH). A selective depletion complex (SDC) comprising a pH-independent receptor-binding peptide and a pH-dependent target-binding peptide may be catalytic (e.g., reused). The SDC may stay bound to the receptor through multiple rounds of endocytosis and has the potential to carry another target molecule in each round and leave the target molecule in the endosome/lysosome for degradation. Thus, one catalytic SDC molecule may cause the degradation of multiple target molecules. [0338] In some embodiments, the receptor-binding peptide can bind to the receptor with an affinity that is pH dependent. For example, the receptor-binding molecule can bind to the receptor with higher affinity at extracellular pH (such as about pH 7.4) and with lower affinity at a lower endosomal pH (such as about pH 5.5, about pH 5.8, or about pH 6.5), thereby releasing the selective depletion complex from the receptor upon internalization and acidification of the endosomal compartment. In some embodiment, the receptor-binding peptide can bind the receptor with an affinity that is pH dependent and the target-binding peptide can bind the target with an affinity that is pH dependent or that is pH-independent. In some embodiments, the selective depletion complex releases the target (or the receptor) in the endosome with fast enough dissociation kinetics that the target (or the target-selective depletion complex complex) is released in the endosome regardless of the effect of pH on binding. A selective depletion molecule can be used to selectively deplete a target molecule (e.g., a soluble protein or a cell surface protein). For example, a selective depletion complex comprising a receptor-binding peptide and a target-binding peptide can bind to the receptor via the receptor-binding peptide and to a target molecule (e.g., a soluble protein or a cell surface protein). The target molecule can be delivered to an endocytic compartment via receptor-mediated endocytosis of the receptor and the selective depletion molecule. In the endocytic compartment, the selective depletion complex can remain bound to the receptor, and the target molecule can be released from the selective depletion complex as the endocytic compartment acidifies. The selective depletion molecule can be recycled to the cell surface along with the receptor, and the target molecule can continue to the lysosome where it is degraded. In some embodiments, the target molecule can remain in the endosome or lysosome without being degraded, resulting in enrichment of the target molecule in the endosome or lysosome. In some embodiments, the selective depletion complexes of the present disclosure can have a low molecular weight compared to targetbinding antibodies and can be used to bind and deplete a target without requiring a supply and distribution cold chain.

[0339] In some embodiments, a receptor-binding peptide (a TfR-binding peptide or a PD-L1- binding peptide) may bind to a cellular receptor (e.g., TfR or PD-L1) with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM. In some embodiments, a receptor-binding peptide (a TfR-binding peptide or a PD-Ll-binding peptide) may bind to a cellular receptor (e.g., TfR or PD-L1) with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM. In some embodiments, a receptor-binding peptide (a TfR-binding peptide or a PD-L1 -binding peptide) may bind to a cellular receptor (e.g., TfR or PD-L1) with a dissociation rate constant (k_off or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹. In some embodiments, a receptor-binding peptide has an off rate that is slower than the recycling rate of the cellular receptor, such that the receptor-binding peptide is likely to remain bound to receptor during the recycling process. In some embodiments, the receptor-binding peptide may have an off rate that is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, or no faster than 20 minutes. In some embodiments, the receptor-binding peptide may have an off rate that is from about 1 minute to about 20 minutes, from about 2 minutes to about 15 minutes, from about 2 minutes to about 10 minutes, or from about 5 minutes to about 10 minutes.

[0340] The selective depletion complexes of the present disclosure can be used to treat a disease or a condition by selectively depleting a target molecule that is associated with the disease or the condition. For example, a selective depletion complex can be used to selectively deplete a soluble or cell surface protein that accumulates, contains a disease-associated mutation (e.g., a mutation causing constitutive activity, resistance to treatment, or dominant negative activity), or is over-expressed in a disease state. In some embodiments, the selective depletion complexes of the present disclosure can be used for the treatment and prevention of various neurological diseases including but not limited to epilepsy, schizophrenia, depression, anxiety, bipolar disorder, developmental brain disorders (e.g., autism spectrum), or mood disorder.

[0341] Binding of the herein described selective depletion complexes (e.g., peptide conjugates, fusion peptides, or recomb inantly produced peptide complexes) to TfR and subsequent transport across a cell layer or barrier such as the BBB (e.g., via vesicular transcytosis) or a cell membrane (e.g., via endocytosis) can have implications in a number of diseases, conditions, or disorders associated with EGFR-driven cancer.

[0342] Binding of the herein described selective depletion complexes (e.g., peptide conjugates, fusion peptides, or recomb inantly produced peptide complexes) to TfR and subsequent transport across a cell layer or barrier such as the BBB (e.g., via vesicular transcytosis) or a cell membrane (e.g., via endocytosis) can have implications in various cancers. Cancers that can treated or prevented with the herein described selective depletion complexes can include breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, blood-cell-derived cancer, spleen cancer, lung cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, esophageal cancer, gastrointestinal (GI) cancers, thyroid cancer, endometrial cancer, bladder cancer, cancers of the salivary gland, kidney cancer, muscle cancers, ovarian cancer, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, bone marrow cell cancers, skin cancer, melanoma, genitourinary cancer, osteosarcoma, muscle- derived sarcoma, melanoma, head and neck cancer, a neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, and diffuse intrinsic pontine glioma (DIPG), a CMY C-overexpressing cancer, primary cancers in the brain, or cancers that have metastasized to the brain. For example, a selective depletion complex comprising a target-binding peptide that binds a protein associated with cancer (e.g., EGFR) can be used to treat a cancer. In some embodiments, a selective depletion complex for treatment of a cancer can comprise a target-binding peptide that binds an extracellular, soluble, or cell surface protein associated with cell growth, cell division, avoidance of cell death, immune evasion, suppression of inflammatory responses, promotion of vascular growth, or protection from hypoxia.

[0343] In some embodiments, a selective depletion complex (e.g., comprising a target-binding peptide and a cellular receptor-binding peptide) or a selective depletion complex component (e.g., comprising a target-binding peptide or a cellular receptor-binding peptide and a dimerization domain capable of facilitating dimerization to form a complete selective depletion complex) of any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526. In some embodiments, an EGFR inhibitor peptide complex (e.g., comprising a target-binding peptide and a dimerization domain capable of dimerizing to form a peptide complex capable of EGFR inhibition) may comprise a sequence of SEQ ID NO: 498 or SEQ ID NO: 502. In some embodiments, a selective depletion complex (e.g., comprising a target-binding peptide and a cellular receptor-binding peptide) or a selective depletion complex component (e.g., comprising a target-binding peptide or a cellular receptor-binding peptide and a dimerization domain capable of facilitating dimerization to form a complete selective depletion complex) may comprise a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526, or a fragment thereof. In some embodiments, an EGFR inhibitor peptide complex (e.g., comprising a target-binding peptide and a dimerization domain capable of dimerizing to form a peptide complex capable of EGFR inhibition) may comprise a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 498 or SEQ ID NO: 502. Examples of selective depletion complexes, complex components, and EGFR inhibitor peptide complex sequences are provided in TABLE 8. In some embodiments, the target-binding peptide portion of the selective depletion complex comprising a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526 may be replaced with an EGFR target-binding peptide of any one of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. In some embodiments, the target-binding peptide portion of the EGFR inhibitor peptide complex comprising a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 498 or SEQ ID NO: 502 may be replaced with an EGFR target-binding peptide of any one of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

TABLE 8 - Selective Depletion Complex (SDC), Complex Components Sequences, and

EGER Inhibitor Complex Sequences

Use of Non-Activating EGF Variants for EGFR Inhibition

[0344] A non-activating EGF variant peptide or peptide complex of the present disclosure (e.g., any one of SEQ ID NO: 388 - SEQ ID NO: 390, SEQ ID NO: 457 - SEQ ID NO: 494, SEQ ID NO: 498, or SEQ ID NO: 502) or a non-activating EGF variant peptide complex may be administered to a subject (e.g., a human or non-human animal subject) to inhibit EGFR activity in the subject. EGFR activity may be associated with uncontrolled cell growth, cell survival, immunosuppression, therapeutic treatment resistance, transcriptional program alteration, or other processes contributing to cancer progression, and inhibiting EGFR activity may reduce cancer cell growth, survival, immunosuppression, therapeutic treatment resistance, transcriptional program alteration, other processes contributing to cancer progression, or a combination thereof. Inhibition of EGFR may be beneficial in diseases such as cancer driven by MEK/ERK and/or PI3K/AKT signaling or other signaling pathways to which EGFR signaling contributes. In some embodiments, inhibiting EGFR (e.g., by administering a non-activating EGF variant peptide) may disrupt EGFR-driven growth and survival pathways, thereby treating the cancer.

[0345] The non-activating EGF variant peptides of the present disclosure may inhibit EGFR by blocking interactions between EGFR and normal EGF. For example, SEQ ID NO: 494 shares its EGFR Domain III binding site with normal EGF, preventing EGF from accessing the binding interface. In some embodiments, the non-activating EGF variant peptides of the present disclosure may inhibit EGFR by binding to EGFR Domain III and preventing normal EGF from contacting both Domain III and Domain I, which is mechanistically known to induce a conformation change in Domain II that permits homo- and hetero-dimerization.

[0346] Administration of a non-activating EGF variant peptide or non-activating EGF variant peptide complex may be used in a method of treating cancer by binding to and inhibiting EGFR upon administration to a subject. Inhibition of EGFR may disrupt cancer cell growth, survival, immunosuppression, therapeutic treatment resistance, transcriptional program alteration, or a combination thereof. The non-activating EGF variant peptides described herein may be used to treat any cancer driven in part by signaling arising from EGFR homo- or heterodimerization. Examples of cancers that may be treated by administering a non-activating EGF variant peptide or non-activating EGF variant peptide complex include melanoma, non-small cell lung cancer, small cell lung cancer, renal cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, lymphoma, bladder cancer, liver cancer, gastric cancer, breast cancer, pancreatic cancer, prostate cancer, Merkel cell carcinoma, mesothelioma, or brain cancer (e.g., glioblastoma, astrocytoma, meningioma, metastatic brain cancer, or primary brain cancer).

Sequence Identity and Homology

[0347] Percent (%) sequence identity or homology is determined by conventional methods. (See e.g., Altschul et al. (1986), Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff (1992), Proc. Natl. Acad. Sci. USA 89:10915). Briefly, two amino acid sequences can be aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (Id.). The sequence identity or homology is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).

[0348] Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. Pairwise sequence alignment can be used to identify regions of similarity that can indicate functional, structural and/or evolutionary relationships between two biological sequences (e.g., amino acid or nucleic acid sequences). In addition, multiple sequence alignment (MSA) is the alignment of three or more biological sequences. From the output of MSA applications, homology can be inferred and the evolutionary relationship between the sequences assessed. As used herein, “sequence homology” and “sequence identity” and “percent (%) sequence identity” and “percent (%) sequence homology” are used interchangeably to mean the sequence relatedness or variation, as appropriate, to a reference polynucleotide or amino acid sequence.

[0349] Additionally, there are several established algorithms available to align two amino acid sequences. For example, the “FASTA” similarity search algorithm of Pearson and Lipman can be a suitable protein alignment method for examining the level of sequence identity or homology shared by an amino acid sequence of a peptide disclosed herein and the amino acid sequence of a peptide variant. The FASTA algorithm is described, for example, by Pearson and Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 1) and a test sequence that has either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score. If there are several regions with scores greater than the “cutoff’ value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. For example, illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183 63 (1990).

[0350] FASTA can also be used to determine the sequence identity or homology of nucleic acid sequences or molecules using a ratio as disclosed above. For nucleic acid sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described herein.

[0351] Some examples of common amino acids that are a “conservative amino acid substitution” are illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat ’I Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language “conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0352] Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that can be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity or homology and computer analysis using available software (e.g., the Insight II.RTM. viewer and homology modeling tools; MSI, San Diego, Calif.), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, G.J., Current Opin. Struct. Biol. 5:372-6 (1995) and Cordes, M.H. et al., Current Opin. Struct. Biol. 6:3-10 (1996)). In general, when designing modifications to molecules or identifying specific fragments, determination of structure can typically be accompanied by evaluating activity of modified molecules.

Engineered Binding Peptides

[0353] A peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, a target-binding peptide such as an EGFR target-binding peptide, or a selective depletion complex) can be engineered to improve or alter a property of the peptide. For example, a peptide can be modified to alter the affinity of the peptide for a binding partner (e.g., an EGFR target molecule or a TfR). In some embodiments, a peptide can be modified to alter binding affinity in a pH-dependent manner. A peptide can be modified my introducing one or more amino acid variations into the peptide sequence and testing the effect of the variation on peptide properties (e.g., binding affinity).

[0354] In some embodiments, a peptide or a library of peptides is designed in silico without derivation from a naturally occurring scaffold of a knotted peptide. In other embodiments, a peptide or a library of peptides is designed in silico by derivation, grafting relevant proteinbinding residues, or conserved residues in the protein-binding interface a naturally occurring peptide or protein known to bind to a protein or receptor of interest. In some embodiments, the peptide (e.g., a TfR-binding peptide of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) is a simple helix-turn-helix. In some embodiments, the helix-turn-helix can be used for pharmacophore transfer onto other scaffolds, for example engraftment of the required TfR-engaging surface onto the helix-tum-helix scaffold using fusion tagging.

[0355] In some embodiments, a peptide comprising SEQ ID NO: 1 is used as a scaffold or base sequence for further modifications, including addition, deletion, or amino acid substitution. In some embodiments, short sequences of amino acid residues such as GS are added at the N- terminus of a peptide. In some embodiments, peptides lack GS at the N-terminus. In some instances, peptides undergo one or more post-translational modifications.

[0356] In some embodiments, a peptide capable of binding TfR and transcytosis across a cell membrane comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the exemplary peptide sequences listed in TABLE 1 (SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64), or a functional fragment thereof. Two or more peptides can share a degree of sequence identity or homology and share similar properties in vivo. For instance, a peptide can share a degree of sequence identity or homology with any one of the peptides of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. In some embodiments, one or more peptides of the present disclosure have up to about 20% pairwise sequence identity or homology, up to about 25% pairwise sequence identity or homology, up to about 30% pairwise sequence identity or homology, up to about 35% pairwise sequence identity or homology, up to about 40% pairwise sequence identity or homology, up to about 45% pairwise sequence identity or homology, up to about 50% pairwise sequence identity or homology, up to about 55% pairwise sequence identity or homology, up to about 60% pairwise sequence identity or homology, up to about 65% pairwise sequence identity or homology, up to about 70% pairwise sequence identity or homology, up to about 75% pairwise sequence identity or homology, up to about 80% pairwise sequence identity or homology, up to about 85% pairwise sequence identity or homology, up to about 90% pairwise sequence identity or homology, up to about 95% pairwise sequence identity or homology, up to about 96% pairwise sequence identity or homology, up to about 97% pairwise sequence identity or homology, up to about 98% pairwise sequence identity or homology, up to about 99% pairwise sequence identity or homology, up to about 99.5% pairwise sequence identity or homology, or up to about 99.9% pairwise sequence identity or homology. In some embodiments, one or more peptides of the disclosure have at least about 20% pairwise sequence identity or homology, at least about 25% pairwise sequence identity or homology, at least about 30% pairwise sequence identity or homology, at least about 35% pairwise sequence identity or homology, at least about 40% pairwise sequence identity or homology, at least about 45% pairwise sequence identity or homology, at least about 50% pairwise sequence identity or homology, at least about 55% pairwise sequence identity or homology, at least about 60% pairwise sequence identity or homology, at least about 65% pairwise sequence identity or homology, at least about 70% pairwise sequence identity or homology, at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85% pairwise sequence identity or homology, at least about 90% pairwise sequence identity or homology, at least about 95% pairwise sequence identity or homology, at least about 96% pairwise sequence identity or homology, at least about 97% pairwise sequence identity or homology, at least about 98% pairwise sequence identity or homology, at least about 99% pairwise sequence identity or homology, at least about 99.5% pairwise sequence identity or homology, at least about 99.9% pairwise sequence identity or homology with a second peptide.

[0357] In some embodiments, peptides that exhibit an improved TfR receptor binding show improved transcytosis function. In some cases, peptides that exhibit an improved TfR receptor binding show no or small changes in transcytosis function. In some cases, peptides that exhibit an improved TfR receptor binding show reduced transcytosis function. In some embodiments, the KA and KD values of a TfR-binding peptide can be modulated and optimized (e.g., via amino acid substitutions) to provide an optimal ratio of TfR-binding affinity and efficient transcytosis function.

[0358] In some instances, the peptide or peptide complex is any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or a functional fragment thereof. In other embodiments, the peptide or peptide complex of the disclosure further comprises a peptide with 99%, 95%, 90%, 85%, or 80% sequence identity or homology to any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 or functional fragment thereof.

[0359] In other instances, the peptide or peptide complex can be a peptide that is homologous to any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or a functional fragment thereof. As further described herein, the term “homologous” can be used herein to denote peptides or peptide complexes having at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95% sequence identity or homology to a sequence of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 or a functional fragment thereof. In various embodiments, a fragment can be least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length. In various embodiments, fragments can be at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids in length. In some embodiments, a fragment can be from about 5 to about 50, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, or from about 10 to about 20 amino acids in length.

[0360] In still other instances, the nucleic acid molecules that encode a peptide or peptide complex of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 can be identified by either a determination of the sequence identity or homology of the encoded peptide amino acid sequence with the amino acid sequence of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or by a nucleic acid hybridization assay. Such peptide variants or peptide complex variants of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x-0.2xSSC with 0.1% SDS at 50-65° C., and (2) that encode a peptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity or homology to the amino acid sequence of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64.

Affinity Maturation

[0361] A peptide of the present disclosure (e.g., a target-binding peptide such as an EGFR target-binding peptide, TfR-b inding peptide, or a selective depletion complex) can be identified or modified through affinity maturation. For example, a target-binding peptide that binds a target of interest can be identified by affinity maturation of a binding peptide (e.g., a CDP, a nanobody, an affibody, a DARPin, a centyrin, a nanofittin, an adnectin, or an antibody fragment). A binding peptide can undergo affinity maturation by generating a library of every possible point mutation, or in the case of a CDP, every possible non-cysteine point mutation. The variant library can be expressed via surface display (e.g., in yeast or mammalian cells) and screened for binding to a binding partner (e.g., an EGFR target molecule or TfR). Library members with increased binding affinity relative to the initial peptide or relative to other members of the variant library can undergo subsequent rounds of maturation. During each round, a variant library of every possible non-cysteine point mutation is generated and screened. In some embodiments, a peptide can undergo 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of affinity maturation to identify a peptide with improved binding affinity to the binding partner of interest (e.g., an EGFR target molecule or TfR). Variants can be identified by Sanger sequencing, next generation sequencing, or high throughput sequencing (e.g., Illumina sequencing).

[0362] In some embodiments, a peptide (e.g., a TfR-binding peptide, a PD-Ll-binding peptide, or an EGFR target-binding peptide) can be selected for pH-independent binding. For example, a peptide can be selected for high affinity binding to a binding partner (e.g., an EGFR target molecule or a TfR) at both extracellular pH (about pH 7.4) and at endosomal pH (such as about pH 5.5). A peptide with pH-independent binding can bind to a binding partner with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at extracellular pH (about pH 7.4). In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at endosomal pH (such as about pH 5.5). In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at endosomal pH (such as about pH 5.8). [0363] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some embodiments, a target-binding peptide with pH- dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.8.

[0364] In some embodiments, the affinity of a target-binding peptide with pH-dependent binding to a target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the affinity of a target-binding peptide with pH-dependent binding to a target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0365] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (k_off or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 7.4. In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’ no more than 5xl0’⁴ s’¹, or no more than 2xl0’⁴ s’¹ at pH 5.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s' or no more than 2x1 O'⁴ s'¹ at pH 5.8.

[0366] In some embodiments, the dissociation rate constant (k_off or kd) of a target-binding peptide with pH-dependent binding to a target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the dissociation rate constant (koff or kd) of a target-binding peptide with pH-dependent binding to a target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0367] In some embodiments the TfR-binding peptides are stable at endosomal pH, and do not release in the endosome for example under acidic conditions, such as pH 6.9, pH 6.8, pH 6.7, pH

6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower.

Conversely, a peptide that has high affinity for binding to a selected target and used in selective depletion complexes as the peptide or peptide complex that binds such selected target molecule and is released in the endosome for degradation within the cell can be a pH-dependent targetbinding CDP such that it is released in the endosome. In some embodiments the target-binding peptides are less stable at endosomal pH, and release wholly or in part in the endosome for example under acidic conditions, such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH

5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. pH-Dependent Binding

[0368] The peptides of the present disclosure (e.g., an EGFR target-binding peptide or a TfR- binding peptide) can be modified for pH-dependent binding properties. Imparting pH-dependent binding to a target-binding peptide (e.g., a target-binding EGF variant) can be performed in three stages. First, a library of peptide variant containing histidine (His) point mutations can be designed. Histidine amino acids are introduced into the target-binding peptide because His is the only natural amino acid whose side chain has a pKa value between neutral (pH 7.4) and acidic (pH <6) endosomal conditions, and this change of charge as pH changes can alter binding, either directly (e.g., changing charge-charge interaction upon formation of a positive charge at low pH) or indirectly (e.g., the change in charge imparts a subtle change in the structure of the targetbinding peptide, disrupting an interface between the target molecule and the target-binding peptide). In some embodiments, a variant screen of the target-binding peptide can be implemented by generating double-His doped libraries. For example, a double-His doped library of a target-binding CDP can comprise a library where every non-Cys, non-His residue is substituted with a His amino acid one- or two-at-a-time. A variant library can be expressed in cells (e.g., yeast cells or mammalian cells) via surface display, with each target-binding peptide variant containing one or two His substitutions. Target-binding peptide variants can be tested for maintenance of binding under neutral pH (about pH 7.4), and for reduced binding under low pH (about pH 6.0, about pH 5.8, or about pH 5.5). Variants that demonstrated reduced binding affinity under low pH as compared to neutral pH can be identified as target-binding peptides with pH-dependent binding. In some embodiments, a pH-dependent EGFR target-binding peptide may comprise one or more His substitutions in a sequence of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

[0369] In some embodiments, the target-binding peptides of the present disclosure (e.g., histidine-containing or histidine-enriched target-binding peptides) can have a high target-binding affinity at physiologic extracellular pH but a significantly reduced binding affinity at lower pH levels such as endosomal pH of 5.5. In some cases, the target-binding peptides of the present disclosure can be optimized for improved intra- vesicular (e.g., intra-endosomal) and/or intracellular delivery function while retaining high target-binding capabilities. In some cases, histidine scans and comparative binding experiments can be performed to develop and screen for such peptides. In some embodiments, an amino acid residue in a peptide of the present disclosure is substituted with a different amino acid residue to alter a pH-dependent binding affinity to a target molecule. The amino acid substitution can increase a binding affinity at low pH, increase a binding affinity at high pH, decrease a binding affinity at low pH, decrease a binding affinity at high pH, or a combination thereof.

[0370] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of less than 50 pM, less than 5 pM, less than 500 nM, less than 100 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM at extracellular pH (such as about pH 7.4). In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, of at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, at least 20 pM, at least 50 pM, at least 100 pM, at least 500 pM, at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 500 mM, or at least 1 M at endosomal pH (such as about pH 5.5). In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, of at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, at least 20 pM, at least 50 pM, at least 100 pM, at least 500 pM, at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least 500 mM, or at least 1 M at endosomal pH (such as about pH 5.8). [0371] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. In some embodiments, a target-binding peptide with pH- dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.8.

[0372] In some embodiments, the affinity of a target-binding peptide with pH-dependent binding can bind a target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the affinity of a target-binding peptide with pH-dependent binding can bind a target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0373] In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (k_off or kd) of no more than 1 s’¹, no more than 5x1 O’¹ s’¹, no more than 2x1 O’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2x1 O’⁴ s’¹ at pH 7.4. In some embodiments, a target-binding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x1 O’¹ s’¹, no more than 2x1 O’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’ no more than 5xl0’⁴ s’¹, or no more than 2xl0’⁴ s’¹ at pH 5.5. In some embodiments, a targetbinding peptide with pH-dependent binding can bind a target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x1 O’¹ s’¹, no more than 2x1 O’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’ or no more than 2x1 O’⁴ s’¹ at pH 5.8.

[0374] In some embodiments, the dissociation rate constant (koff or kd) of a target-binding peptide with pH-dependent binding can bind a target molecule at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the dissociation rate constant (koff or kd) of a target-binding peptide with pH-dependent binding can bind a target molecule at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

[0375] In some embodiments the TfR-binding peptides are stable at endosomal pH, and do not release in the endosome for example under acidic conditions, such as pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. Conversely, a peptide that has high affinity for binding to a selected target and used in selective depletion complexes as the peptide or peptide complex that binds such selected target and is released in the endosome for degradation within the cell can be a pH-dependent target-binding CDP such that it is released in the endosome. In some embodiments the target-binding peptides are less stable at endosomal pH, and release wholly or in part in the endosome for example under acidic conditions, such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower.

Methods of Using Selective Depletion Complexes

[0376] The selective depletion complexes of the present disclosure may be used to exert an effect on a cell, tissue, or subject. The effect may be a therapeutic, pharmacological, biological, or biochemical effect. In some embodiments, the effect may result from selective depletion of a target molecule to which the selective depletion complex binds. In some embodiments, the effect may result from ternary complex formation between a target molecule, a receptor, and a selective depletion complex that binds the target molecule and the receptor.

Selective Depletion of Target Molecules

[0377] Described herein are methods of selectively depleting a target molecule using a composition of the present disclosure (e.g., a selective depletion complex). In some embodiments, a method of the present disclosure can comprise selectively recruiting a molecule to an endocytic compartment via transferrin receptor-mediated endocytosis and enriching the target molecule in the lysosome. In some embodiments, a method of the present disclosure can comprise selectively depleting a molecule from the external environment or the cell surface. In some embodiments, a method of the present disclosure can comprise selectively depleting a molecule from the external environment or the cell surface via transferrin receptor-mediated endocytosis. A selective depletion complex (e.g., a peptide complex comprising a receptorbinding peptide conjugated to a target-binding peptide such as an EGFR target-binding peptide) can bind to the receptor via the receptor-binding peptide and to a target molecule (e.g., a soluble protein, an extracellular protein, or a cell surface protein). The target molecule can be delivered to an endocytic compartment via receptor-mediated endocytosis of the receptor and the selective depletion molecule. In the endocytic compartment, the selective depletion complex can remain bound to the receptor, and the target molecule can be released from the selective depletion complex as the endocytic compartment acidifies. In some embodiments, the selective depletion molecule can be recycled to the cell surface along with the receptor, and the target molecule can continue to the lysosome where it is degraded. In some embodiments, the target molecule can remain in the endosome or the lysosome without being degraded, resulting in enrichment of the target molecule in the endosome or the lysosome, such as lysosomal enzymes in lysosomal storage diseases.

[0378] The methods of the present disclosure for selectively depleting a target molecule (e.g., an EGFR target molecule) or for selectively enriching a target molecule in the lysosome can be used to treat a disease or condition associated with the target molecule. For example, selective depletion of a target molecule associated with cancer can be used to treat the cancer. Depletion of a cell surface molecule can allow the cancer cell to be targeted by the immune system, to lose checkpoint inhibition, can disable survival signaling, or remove drug resistance pumps. In another example, selective enrichment in the lysosome of a lysosomal enzyme associated with a lysosomal storage disease can be used to treat the lysosomal storage disease. In this example, a lysosomal enzyme can be administered in co-therapy with the target-depleting complex, such that the target depleting complex drives the lysosomal enzyme into the lysosomal compartment. A method of treating a disease or condition can comprise contacting a cell (e.g., a cell expressing the receptor) with a selective depletion complex of the present disclosure. In some embodiments, the selective depletion complex can be administered to a subject (e.g., a human subject) having a disease or condition (e.g., a neurodegenerative disease, a cancer, harmful inflammation, or a lysosomal storage disease).

[0379] TfR is a fairly ubiquitous protein, as all mammalian cells require iron and therefore take up transferrin through this constitutive pathway. By this mechanism, virtually any target tissue would be amenable to the selective depletion methods or selective enrichment methods of the present disclosure comprising a TfR-binding peptide. Tumor tissue can be particularly well- suited for the methods of the present disclosure as most tumors are enriched for TfR, which can impart natural tumor selectivity in the selective depletion molecules. TfR has been identified as a potential universal cancer marker. Tumors promoting angiogenesis can also overexpress TfR, as both vascular endothelial growth factor (VEGF) and TfR can be expressed as a result of hypoxia-inducible factor (HIF-la)-driven transcriptional programs, and thus be a favorable tissue for selective depletion methods involving transferrin receptor-mediated use of SDCs described herein.

[0380] Liver tissue can also be highly enriched for TfR and thus be a favorable tissue for selective depletion methods. In some embodiments, the selective depletion complexes of the present disclosure (e.g., selective depletion complexes comprising a CDP) can be stable in the liver for extended periods of time. For example, a selective depletion complex of the present disclosure can have a half-life in the liver of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 hours. Serum proteins, which can already largely be subject to hepatic metabolism as a class, could be targeted for selective depletion with relatively low doses of selective depletion complexes. Serum half-life of the selective depletion complexes of the present disclosure could be improved to create a molecule that requires infrequent dosing, for example by addition of a serum half-life extension peptide. Selective depletion complexes with a shorter half-life can serve as an acute target elimination drug, for example to treat harmful inflammatory signaling.

[0381] A selective depletion complex can be administered to a subject systemically or peripherally and can accumulate in tissue with high levels of TfR expression (e.g., tumor tissue, kidney tissue, spleen, bone marrow, or liver tissue) or high levels of target expression or with high levels of both receptor (e.g., TfR) and target. In some embodiments, a selective depletion complex can be administered to a subject systemically or peripherally and can accumulate in tumor tissue, kidney tissue, or liver tissue. In some embodiments, a selective depletion complex can comprise a tissue targeting domain and can accumulate in the target tissue upon administration to a subject. For example, selective depletion complexes can be conjugated to, linked to, or fused to a molecule (e.g., small molecule, peptide, or protein) with targeting or homing function for a cell of interest or a target protein located on the surface or inside said cell. In some embodiments, a selective depletion complex can be administered to a subject orally and can reach the gastrointestinal tract. Orally administered selective depletion complexes can be used for clearance of disease-associated proteins in the gastrointestinal tract.

[0382] In some embodiments, a selective depletion complex of the present disclosure can be genetically encoded into a benign cell with a secretory phenotype. The selective depletion complex can be expressed by the secretory cell and administered as a secreted molecule in a localized cellular therapy. In some embodiments, a gene encoding a selective depletion complex can be delivered as a gene therapy to a tissue of interest (e.g., liver, hematopoietic, kidney, skin, tumor, central nervous system (CNS), or neurons).

[0383] In some embodiments, an EGF variant peptide construct may comprise a miniprotein, a nanobody, an antibody, an IgG, an antibody fragment, a Fab, a F(ab)2, an scFv, an (scFv)2, a DARPin, or an afflbody. In some embodiments, the target-binding peptide may comprise a cystine-dense peptide, an affltin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some embodiments, the target molecule is over-expressed in the disease or condition and depleting the target molecule reduces the level of the target molecule, thereby treating the disease or condition. In some embodiments, the target molecule accumulates in the disease or condition and depleting the target molecule clears or reduces the accumulation, thereby treating the disease or condition. In some embodiments, the target molecule is hyper-activated or over-stimulated, and depleting the target molecule reduces a level of activity of the target molecule, thereby treating the disease or condition. Examples of diseases that may be treated using a selective depletion complex include cancers, (e.g., non-small-cell lung cancer, primary non-small-cell lung cancer, metastatic non- small-cell lung cancer, head and neck cancer, head and neck squamous cell carcinoma, glioblastoma, brain cancer, metastatic brain cancer, colorectal cancer, colon cancer, tyrosine kinase inhibitor (TKI)-resistant cancer, cetuximab-resistant cancer, necitumumab-resistant cancer, panitumumab-resistant cancer, local cancer, regionally advanced cancer, recurrent cancer, metastatic cancer, refractory cancer, KRAS wildtype cancer, KRAS mutant cancers, or exon20 mutant non-small-cell lung cancer). In some embodiments, the cancer has one or more of the following: overexpresses EGFR, KRAS mutation, KRAS G12S mutation, KRAS G12C mutation, PTEN loss, EGFR exonl9 deletion, EGFR L858R mutation, EGFR T790M mutation, a cetuximab-resistant EGFR, a panitumumab-resistant EGFR, PIK3CA mutation, TP53 R273H mutation, PIK3CA amplification, PIK3CA G118D, TP53 R273H, EGFR C797X mutation, EGFR G724S mutation, EGFR L718Q mutation, EGFR S768I mutation, an EGFR mutation, or a combination thereof. In some embodiments, the cancer expresses or has upregulated c-MET, Her2, Her3 that heterodimerizes with EGFR.

[0384] Administration of a selective depletion complex of the present disclosure may be combined with an additional therapy to treat a disease or condition. In some embodiments, the additional therapy is adjuvant, first-line, or combination therapy. In some embodiments, the additional therapy comprises radiation, chemotherapy, platinum therapy, anti-metabolic therapy, targeted therapy to other oncogenic signaling pathways, targeted therapy to immune response pathways, therapy aimed at directly driving an immune response to cancer cells, or targeted therapies disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. In some embodiments, the targeted therapy to other oncogenic signaling pathways comprises administration of inhibitors of MEK/ERK pathway signaling, PI3K/AKT pathway signaling, JAK/STAT pathway signaling, or WNT/p-catenin pathway signaling. In some embodiments, the targeted therapy to immune response pathways comprises PD-1/PD-L1 checkpoint inhibition. In some embodiments, the therapy aimed at therapy aimed at directly driving an immune response to cancer cells comprises bispecific T cell engagers or chimeric antigen receptor expressing T cells. In some embodiments, the targeted therapies disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells comprises administering seno lytic agents to a subject. For example, administration of a selective depletion complex to treat a cancer may be combined with administration of radiation therapy, chemotherapy, platinum therapy, or anti-metabolic therapy. In some embodiments, the additional therapy may comprise administering fluorouracil, FOLFIRI, irinotecan, FOLFOX, gemcitabine, cisplatin, irinotecan, oxiplatin, or fluoropyrimidine to the subject.

Ternary Complex Formation

[0385] Described herein are methods of forming a ternary complex between a target molecule, a receptor, and a selective depletion complex comprising a receptor-binding peptide and a targetbinding peptide. The ternary complex may form through binding of the receptor-binding peptide to the receptor and binding of the target-binding peptide to the target. Ternary complex formation between the target, the receptor, and the selective depletion complex may exert a therapeutic, pharmacological, biological, or biochemical effect on a cell, tissue, or subject expressing the target and the receptor. In some embodiments, formation of a ternary complex between a receptor, a target, and a selective depletion complex may increase recycling or turnover of the target molecule, the receptor, or both. Increased recycling or turnover of the target or the receptor may alter (e.g., increase) activity of the target or the receptor, thereby exerting a therapeutic, pharmacological, biological, or biochemical effect.

[0386] Formation of the ternary complex may exert a therapeutic, pharmacological, biological, or biochemical by recruiting the target molecule to the receptor. Recruitment of the target molecule to the receptor may promote a binding interaction between the receptor and the target. In some embodiments, subsequent recycling of the receptor and the target may facilitate the therapeutic, pharmacological, biological, or biochemical effect. In some embodiments, formation of the ternary complex may increase, facilitate, or stabilize the interaction between the target and the receptor.

[0387] In some embodiments, peptide complexes comprising one or more EGFR-binding peptides (e.g., target-binding EGF variants) as described herein conjugated to, linked to, or fused to, or complexed with one or more active agents (e.g., therapeutic agents, detectable agents, diagnostic, contrast, stabilizing agent, or other agent), or combinations thereof. Active agents that may be complexed with or administered with a target-binding peptide (e.g., a target-binding EGF variant or an SDC) may comprise a peptide (e.g., an oligopeptide or a polypeptide), a peptidomimetic, an oligonucleotide, a DNA (e.g., cDNA, ssDNA, or dsDNA), an RNA (e.g., an RNAi, microRNA, snRNA, dsRNA, or antisense oligonucleotide), an antibody, a single chain variable fragment (scFv), an antibody fragment, nanobody, an aptamer, or a small molecule. In some embodiments, an EGF variant peptide may comprise a miniprotein, a nanobody, an antibody, an IgG, an antibody fragment, a Fab, a F(ab)2, an scFv, an (scFv)2, a DARPin, or an afflbody. In some embodiments, the target-binding peptide may comprise a cystine-dense peptide, an affltin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. In some embodiments, the active agent may be an anti-cancer agent. Examples of anti-cancer agents include radionuclides, radioisotopes, chemotherapeutic agents, platinum therapeutics, toxins, enzymes, sensitizing drugs, nucleic acids, including interfering RNAs, antibodies, anti-angiogenic agents, cisplatin, anti-metabolites, anti-metabolic therapeutics, mitotic inhibitors, growth factor inhibitors, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, amifostine, and their equivalents. An active agent may have an anti- metabolic effect, target oncogenic signaling pathways, target immune response pathways, directly drive an immune response to cancer cells, or target disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. A peptide construct of the present disclosure can comprise an EGFR-binding peptide (e.g., an EGF variant of any of SEQ ID NO: 314, SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494), that is linked to one or more active agents via one or more linker moieties (e.g., cleavable or stable linker) as described herein (e.g., a linker of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541) or a half-life extending peptide (e.g., SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 192, SEQ ID NO: 245 - SEQ ID NO: 287, SEQ ID NO: 535 - SEQ ID NO: 537, or SEQ ID NO: 706 - SEQ ID NO: 710).

[0388] An EGFR-binding peptide may be complexed with a detectable agent that comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, a radioisotope, nanoparticle, a paramagnetic metal ion, or a combination thereof. The compounds and methods of the present disclosure can be used alone or in combination with a companion diagnostic, therapeutic or imaging agent (whether such diagnostic, therapeutic or imaging agent is a fluorophore alone, or conjugated, fused, linked, or otherwise attached to a chemical agent or other moiety, small molecule, therapeutic, drug, protein, peptide, antibody protein or fragment of the foregoing, and in any combination of the foregoing; or used as a separate companion diagnostic, therapeutic or imaging agent in conjunction with the fluorophore or other detectable moiety is alone, conjugated, fused, linked, or otherwise attached to a chemical agent or other moiety, small molecule, therapeutic, drug, peptide, antibody protein or fragment of the foregoing, and in any combination of the foregoing). Such companion diagnostics can utilize agents including chemical agents, radiolabel agents, radiosensitizing agents, fluorophores, imaging agents, diagnostic agents, protein, peptide, or small molecule such agent intended for or having diagnostic or imaging effect. Agents used for companion diagnostic agents and companion imaging agents, and therapeutic agents, can include the diagnostic, therapeutic and imaging agents described herein or other known agents. Diagnostic tests can be used to enhance the use of therapeutic products, such as those disclosed herein or other known agents. The development of therapeutic products with a corresponding diagnostic test, such as a test that uses diagnostic imaging (whether in vivo, ex vivo or in vitro) can aid in diagnosis, treatment, identify patient populations for treatment, and enhance therapeutic effect of the corresponding therapy. The compounds and methods of the present disclosure can also be used to detect therapeutic products, such as those disclosed herein or other known agents, to aid in the application of a therapy and to measure it to assess the agent’s safety and physiologic effect, e.g. to measure bioavailability, uptake, distribution and clearance, metabolism, pharmacokinetics, localization, blood concentration, tissue concentration, ratio, measurement of concentrations in blood and/or tissues, assessing therapeutic window, extending visibility window, range and optimization, and the like of the therapeutic agent. Such The compounds and methods can be employed in the context of therapeutic, imaging and diagnostic applications of such agents. Tests also aid therapeutic product development to obtain the data FDA uses to make regulatory determinations. For example, such a test can identify appropriate subpopulations for treatment or identify populations who should not receive a particular treatment because of an increased risk of a serious side effect, making it possible to individualize, or personalize, medical therapy by identifying patients who are most likely to respond, or who are at varying degrees of risk for a particular side effect. Thus, the present disclosure, in some embodiments, includes the joint development of therapeutic products and diagnostic devices, including the compounds and methods herein (used to detect the therapeutic and/or imaging agents themselves, or used to detect the companion diagnostic or imaging agent, whether such diagnostic or imaging agent is linked to the therapeutic and/or imaging agents or used as a separate companion diagnostic or imaging agent linked to the peptide for use in conjunction with the therapeutic and/or imaging agents) that are used in conjunction with safe and effective use of the therapeutic and/or imaging agents as therapeutic or imaging products. Non-limiting examples of companion devices include a surgical instrument, such as an operating microscope, confocal microscope, fluorescence scope, exoscope, endoscope, or a surgical robot and devices used in biological diagnosis or imaging or that incorporate radiology, including the imaging technologies of X-ray radiography, magnetic resonance imaging (MRI), medical ultrasonography or ultrasound, endoscopy, elastography, tactile imaging, thermography, medical photography and nuclear medicine functional imaging techniques as positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Companion diagnostics and devices can comprise tests that are conducted ex vivo, including detection of signal from tissues or cells that are removed following administration of the companion diagnostic to the subject, or application of the companion diagnostic or companion imaging agent directly to tissues or cells following their removal from the subject and then detecting signal.

Physicochemical Properties of Peptides

[0389] In some embodiments, a peptide of the present disclosure (e.g., a TfR-binding peptide, a PD-L1 -binding peptide, a target-binding peptide such as an EGFR target-binding peptide, or a selective depletion complex) can comprise a wide range of physicochemical properties such as molecular size and structure, pH, isoelectric point, and overall molecular net charge. These parameters can have an effect on the peptides ability to bind TfR, bind a target molecule (e.g., an EGFR target molecule), promote transcytosis, transport of cargo molecules across cell barrier such as the BBB, or combinations thereof.

[0390] A peptide of the present disclosure can comprise at least one amino acid residue in D configuration. In some embodiments, a peptide is about 5-100 amino acid residues long. In some embodiments, a peptide is about 10-90 amino acid residues long. In some embodiments, a peptide is about 15-80 amino acid residues long. In some embodiments, a peptide is about 15-75 amino acid residues long. In some embodiments, a peptide is about 15-70 amino acid residues long. In some embodiments, a peptide is about 20-65 amino acid residues long. In some embodiments, a peptide is about 20-60 amino acid residues long. In some embodiments, a peptide is about 25-55 amino acid residues long. In some embodiments, a peptide is about 25-50 amino acid residues long. In some embodiments, a peptide is about 25-40 amino acid residues long. In some embodiments, a peptide is about 11-35 amino acid residues long. In some embodiments, a peptide is about 10-25 amino acid residues long.

[0391] In some embodiments, a peptide is at least 5 amino acid residues long. In some embodiments, a peptide is at least 10 amino acid residues long. In some embodiments, a peptide is at least 15 amino acid residues long. In some embodiments, a peptide is at least 20 amino acid residues long. In some embodiments, a peptide is at least 25 amino acid residues long. In some embodiments, a peptide is at least 30 amino acid residues long. In some embodiments, a peptide is at least 35 amino acid residues long. In some embodiments, a peptide is at least 40 amino acid residues long. In some embodiments, a peptide is at least 45 amino acid residues long. In some embodiments, a peptide is at least 50 amino acid residues long. In some embodiments, a peptide is at least 55 amino acid residues long. In some embodiments, a peptide is at least 60 amino acid residues long. In some embodiments, a peptide is at least 65 amino acid residues long. In some embodiments, a peptide is at least 70 amino acid residues long. In some embodiments, a peptide is at least 75 amino acid residues long.

[0392] In some embodiments, an amino acid sequence of a peptide as described herein comprises at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, or at least 81 amino acid residues.

[0393] In some embodiments of the present disclosure, a three-dimensional or tertiary structure of a peptide is primarily comprised of beta-sheets and/or alpha-helix structures. In some embodiments, designed or engineered peptides (e.g., target-binding peptides, TfR-binding peptides, or selective depletion complexes) of the present disclosure are small, compact peptides or polypeptides stabilized by intra-chain disulfide bonds (e.g., mediated by cysteines) and a hydrophobic core. In some embodiments, engineered peptides have structures comprising helical bundles with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptides. In other embodiments, the engineered TfR-b inding peptides comprise structures with three alpha helices and three intra-chain disulfide bonds, one between each of the three alpha helices in the bundle of alpha helices.

[0394] At physiologic extracellular pH, peptides as described herein can have an overall molecular net charge, for example, of -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, or +5. When the net charge is zero, the peptide can be uncharged or zwitterionic. In some embodiments, a peptide contains one or more disulfide bonds and has a positive net charge at physiologic extracellular pH where the net charge can be +0.5 or less than +0.5, +1 or less than +1, +1.5 or less than +1.5, +2 or less than +2, +2.5 or less than +2.5, +3 or less than +3, +3.5 or less than +3.5, +4 or less than +4, +4.5 or less than +4.5, +5 or less than +5, +5.5 or less than +5.5, +6 or less than +6, +6.5 or less than +6.5, +7 or less than +7, +7.5 or less than +7.5, +8 or less than +8, +8.5 or less than +8.5, +9 or less than +9.5, +10 or less than +10. In some embodiments, a peptide has a negative net charge at physiologic extracellular pH where the net charge can be -0.5 or less than -0.5, -1 or less than -1, -1.5 or less than -1.5, -2 or less than -2, -2.5 or less than -2.5, -3 or less than -3, -3.5 or less than -3.5, -4 or less than -4, -4.5 or less than -4.5, -5 or less than -5, -5.5 or less than -5.5, -6 or less than -6, -6.5 or less than -6.5, -7 or less than -7, -7.5 or less than -7.5, -8 or less than -8, -8.5 or less than -8.5, -9 or less than -9.5, -10 or less than -10.

[0395] In some embodiments, peptides of the present disclosure can have an isoelectric point (pl) value from 3 and 10. In other embodiments, peptides of the present disclosure can have a pl value from 4.3 and 8.9. In some embodiments, peptides of the present disclosure can have a pl value from 3-4. In some embodiments, peptides of the present disclosure can have a pl value from 3-5. In some embodiments, peptides of the present disclosure can have a pl value from 3-6. In some embodiments, peptides of the present disclosure can have a pl value from 3-7. In some embodiments, peptides of the present disclosure can have a pl value from 3-8. In some embodiments, peptides of the present disclosure can have a pl value from 3-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-5. In some embodiments, peptides of the present disclosure can have a pl value from 4-6. In some embodiments, peptides of the present disclosure can have a pl value from 4-7. In some embodiments, peptides of the present disclosure can have a pl value from 4-8. In some embodiments, peptides of the present disclosure can have a pl value from 4-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-10. In some embodiments, peptides of the present disclosure can have a pl value from 5-6. In some embodiments, peptides of the present disclosure can have a pl value from 5-7. In some embodiments, peptides of the present disclosure can have a pl value from 5-8. In some embodiments, peptides of the present disclosure can have a pl value from 5-9. In some embodiments, peptides of the present disclosure can have a pl value from 5-10. In some embodiments, peptides of the present disclosure can have a pl value from 6-7. In some embodiments, peptides of the present disclosure can have a pl value from 6-8. In some embodiments, peptides of the present disclosure can have a pl value from 6-9. In some embodiments, peptides of the present disclosure can have a pl value from 6-10. In some embodiments, peptides of the present disclosure can have a pl value from 7-8. In some embodiments, peptides of the present disclosure can have a pl value from 7-9. In some embodiments, peptides of the present disclosure can have a pl value from 7-10. In some embodiments, peptides of the present disclosure can have a pl value from 8-9. In some embodiments, peptides of the present disclosure can have a pl value from 8-10. In some embodiments, peptides of the present disclosure can have a pl value from 9-10.

[0396] In some cases, the engineering of one or more mutations within a peptide of the present disclosure (e.g., a TfR-binding peptide) yields a peptide with an altered isoelectric point, charge, surface charge, or rheology at physiologic extracellular pH. Such engineering of a mutation to a peptide that can be derived from a scorpion or spider complex can change the net charge of the peptide, for example, by decreasing the net charge by 1, 2, 3, 4, or 5, or by increasing the net charge by 1, 2, 3, 4, or 5. In such cases, the engineered mutation can facilitate the ability of the peptide to bind a target protein, promote transcytosis, and penetrate a cell, an endosome, or the nucleus. Suitable amino acid modifications for improving the rheology and potency of a peptide can include conservative or non-conservative mutations.

[0397] A peptide can comprise at most 1 amino acid mutation, at most 2 amino acid mutations, at most 3 amino acid mutations, at most 4 amino acid mutations, at most 5 amino acid mutations, at most 6 amino acid mutations, at most 7 amino acid mutations, at most 8 amino acid mutations, at most 9 amino acid mutations, at most 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from. In other embodiments, a peptide, or a functional fragment thereof, comprises at least 1 amino acid mutation, at least 2 amino acid mutations, at least 3 amino acid mutations, at least 4 amino acid mutations, at least 5 amino acid mutations, at least 6 amino acid mutations, at least 7 amino acid mutations, at least 8 amino acid mutations, at least 9 amino acid mutations, at least 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from. In some embodiments, mutations can be engineered within a peptide to provide a peptide that has a desired charge or stability at physiologic extracellular pH.

[0398] A peptide of the current disclosure may have a binding affinity to a molecule (e.g., a target molecule or cellular receptor. The binding affinity may be measured as an equilibrium dissociation constant (KD), a dissociation rate constant (koff or kd), or an off rate (k_off). A dissociation constant (KD) may be no more than 500 nM, no more than 200 nM, 100 nM, no more than 50 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 1 nM, or no more than 0.1 nM. A dissociation rate constant (koff or kd) may be no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5x10’⁵ s’¹, or no more than 2x10’⁵ s’¹. A lower equilibrium dissociation constant (KD) corresponds to a higher affinity (e.g., a higher binding affinity).

[0399] Generally, the nuclear magnetic resonance (NMR)solution structures, the X-ray crystal structures, as well as the primary structure sequence alignment of related structural peptide or protein homologs or in silico design can be used to generate mutational strategies that can improve the folding, stability, and/or manufacturability, while maintaining a particular biological function (e.g., TfR affinity/b inding). A general strategy for producing homologs or in silico designed peptides or polypeptides can include identification of a charged surface patch or conserved residues of a protein, mutation of critical amino acid positions and loops, followed by in vitro and in vivo testing of the peptides. The overall peptide optimization process can be of iterative nature to the extent that, for example, information obtained during in vitro or in vivo testing is used for the design of the next generation of peptides. Hence, the herein disclosed methods can be used to design peptides with improved properties or to correct deleterious mutations that complicate folding and manufacturability. Key amino acid positions and loops can be retained while other residues in the peptide sequences can be mutated to improve, change, remove, or otherwise modify function, such as binding, transcytosis, or the ability to penetrate a cell, endosome, or nucleus in a cell, homing, or another activity of the peptide. These techniques can be used to predict the 3D pharmacophore of a group of structurally homologous scaffolds, as wells as to predict possible graft regions of related proteins to create chimeras with improved properties (e.g., binding properties). For example, this strategy is used to identify critical amino acid positions and loops that are used to design peptides with improved TfR receptor binding and transcytosis properties, high expression, high stability in vivo, or any combination of these properties.

[0400] The present disclosure also encompasses multimers of the various peptides described herein. Examples of multimers include dimers, trimers, tetramers, pentamers, hexamers, heptamers, and so on. A multimer can be a homomer formed from a plurality of identical subunits or a heteromer formed from a plurality of different subunits. In some embodiments, a peptide of the present disclosure is arranged in a multimeric structure with at least one other peptide, or two, three, four, five, six, seven, eight, nine, ten, or more other peptides. In certain embodiments, the peptides of a multimeric structure each have the same sequence. In other embodiments, one or more or all of the peptides of a multimeric structure have different sequences.

[0401] In some embodiments, the present disclosure provides peptide scaffolds that can be used as a starting point for generating additional, next-generation peptides with more specific or improved properties. In some embodiments, these scaffolds are derived from a variety of CDPs or knotted peptides. Some suitable peptides for scaffolds can include, but are not limited to, chlorotoxin, brazzein, circulin, stecrisp, hanatoxin, midkine, hefutoxin, potato carboxypeptidase inhibitor, bubble protein, attractin, a-GI, a-GID, p-PIIIA, co-MVIIA, co-CVID, y-MrlA, p-TIA, conantokin G, contulakin G, GsMTx4, margatoxin, shK, toxin K, chymotrypsin inhibitor (CTI), and EGF epiregulin core. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids can confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.

Pharmacokinetics of Peptides

[0402] The pharmacokinetics of any of the peptides of the present disclosure can be determined after administration of the peptide via different routes of administration. For example, the pharmacokinetic parameters of a peptide of this disclosure can be quantified after intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intranasal, peritoneal, buccal, synovial, intratumoral, or topical administration. Peptides of the present disclosure can be analyzed by using tracking agents such as radiolabels or fluorophores. For example, radiolabeled peptides of this disclosure can be administered via various routes of administration. Peptide concentration or dose recovery in various biological samples such as plasma, urine, feces, any organ, skin, muscle, and other tissues can be determined using a range of methods including HPLC, fluorescence detection techniques (TECAN quantification, flow cytometry, iVIS), or liquid scintillation counting.

[0403] The methods and compositions described herein relate to pharmacokinetics of peptide administration via any route to a subject. Pharmacokinetics can be described using methods and models, for example, compartmental models or non-compartmental methods. Compartmental models include but are not limited to monocompartmental model, the two compartmental model, the multicompartmental model or the like. Models are often divided into different compartments and can be described by the corresponding scheme. For example, one scheme is the absorption, distribution, metabolism and excretion (ADME) scheme. For another example, another scheme is the liberation, absorption, distribution, metabolism and excretion (LADME) scheme. In some aspects, metabolism and excretion can be grouped into one compartment referred to as the elimination compartment. For example, liberation includes liberation of the active portion of the composition from the delivery system, absorption includes absorption of the active portion of the composition by the subject, distribution includes distribution of the composition through the blood plasma and to different tissues, metabolism, which includes metabolism or inactivation of the composition and finally excretion, which includes excretion or elimination of the composition or the products of metabolism of the composition. Compositions administered intravenously to a subject can be subject to multiphasic pharmacokinetic profiles, which can include but are not limited to aspects of tissue distribution and metabolism/excretion. As such, the decrease in plasma or serum concentration of the composition is often biphasic, including, for example an alpha phase and a beta phase, occasionally a gamma, delta or other phase is observed.

[0404] Pharmacokinetics includes determining at least one parameter associated with administration of a peptide to a subject. In some aspects, parameters include at least the dose (D), dosing interval (T), area under curve (AUC), maximum concentration (C_max), minimum concentration reached before a subsequent dose is administered (C_min), minimum time (T_min), maximum time to reach Cmax (T_max), volume of distribution (Vd), steady-state volume of distribution (V_ss), back-extrapolated concentration at time 0 (Co), steady state concentration (Css), elimination rate constant (k_e), infusion rate (kin), clearance (CL), bioavailability (f), fluctuation (%PTF) and elimination half-life (ti/2).

[0405] In certain embodiments, the peptides or peptide complexes of any of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 exhibit optimal pharmacokinetic parameters after oral administration. In other embodiments, the peptides or peptide complexes of any of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 exhibit optimal pharmacokinetic parameters after any route of administration, such as oral administration, inhalation, intranasal administration, topical administration, intravenous administration, subcutaneous administration, intra-articular administration, intramuscular administration, intraperitoneal administration, intra- synovial, or any combination thereof.

[0406] In some embodiments, any peptide or peptide complex of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 exhibits an average Truax of 0.5 - 12 hours, or 1-48 hours at which the Cmax is reached, an average bioavailability in serum of 0.1% - 10% in the subject after administering the peptide to the subject by an oral route, an average bioavailability in serum of less than 0.1% after oral administration to a subject for delivery to the GI tract, an average bioavailability in serum of 10-100% after parenteral administration, an average t>/₂ of 0.1 hours - 168 hours, or 0.25 hours - 48 hours in a subject after administering the peptide to the subject, an average clearance (CL) of 0.5-100 L/hour or 0.5 - 50 L/hour of the peptide after administering the peptide to a subject, an average volume of distribution (Vd) of 200 - 20,000 mL in the subject after systemically administering the peptide to the subject, or optionally no systemic uptake, any combination thereof.

Peptide Stability

[0407] A peptide of the present disclosure can be stable in various biological or physiological conditions, such as physiologic extracellular pH, endosomal or lysosomal pH, or reducing environments inside a cell, in the cytosol, in a cell nucleus, or endosome or a tumor. For example, any peptide or peptide complex comprising any of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64 can exhibit resistance to reducing agents, proteases, oxidative conditions, or acidic conditions.

[0408] In some cases, biologic molecules (such as peptides and proteins) can provide therapeutic functions, but such therapeutic functions are decreased or impeded by instability caused by the in vivo environment. (Moroz et al. Adv Drug Deliv Rev 101:108-21 (2016), Mitragotri et al. Nat Rev Drug Dis cov 13(9):655-72 (2014), Bruno et al. Ther Deliv (11): 1443- 67 (2013), Sinha et al. Crit Rev Ther Drug Carrier Syst. 24(l):63-92 (2007), Hamman et al. BioDrugs 19(3): 165-77 (2005)). For instance, the GI tract can contain a region of low pH (e.g., pH ~1), a reducing environment, or a protease-rich environment that can degrade peptides and proteins. Proteolytic activity in other areas of the body, such as the mouth, eye, lung, intranasal cavity, joint, skin, vaginal tract, mucous membranes, and serum, can also be an obstacle to the delivery of functionally active peptides and polypeptides. Additionally, the half-life of peptides in serum can be very short, in part due to proteases, such that the peptide can be degraded too quickly to have a lasting therapeutic effect when administering reasonable dosing regimens. Likewise, proteolytic activity in cellular compartments such as lysosomes and reduction activity in lysosomes and the cytosol can degrade peptides and proteins such that they can be unable to provide a therapeutic function on intracellular targets. Therefore, peptides that are resistant to reducing agents, proteases, and low pH can be able to provide enhanced therapeutic effects or enhance the therapeutic efficacy of co-formulated or conjugated, linked, or fused active agents in vivo.

Methods of Manufacture

[0409] Various expression vector/host systems can be utilized for the recombinant expression of peptides described herein. Non-limiting examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a nucleic acid sequence encoding peptides, peptide complexes, or peptide fusion proteins/chimeric proteins described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems (including CHO and HEK293 cells) infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus, lentivirus) or transiently or stably transfected with recombinant mammalian expression vectors, including cell lines engineered to contain multiple copies of the aforementioned nucleic acid sequence, either stably amplified (e.g., CHO/dhfr, CHO/glutamine synthetase) or unstably amplified in double-minute chromosomes (e.g., murine cell lines). Disulfide bond formation and folding of the peptide could occur during expression or after expression or both.

[0410] A host cell can be adapted to express one or more peptides described herein. The host cells can be prokaryotic, eukaryotic, or insect cells. In some cases, host cells are capable of modulating the expression of the inserted sequences or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters). In some cases, modifications (e.g., phosphorylation) and processing (e.g., cleavage) of peptide products can be important for the function of the peptide. Host cells can have characteristic and specific mechanisms for the post-translational processing and modification of a peptide. In some cases, the host cells used to express the peptides secrete minimal amounts of proteolytic enzymes.

[0411] The selective depletion complexes of this disclosure can be advantageously made by a single recombinant expression system, with no need for chemical synthesis or modifications. For example, a selective depletion complex can be expressed in CHO cells, HEK cells, yeast, pichia, E. coli, or other organisms. The selective depletion complex may be expressed within the cells and require cell lysis to isolate, or the selective depletion complex may be expressed with trafficking sequences driving secretion from the cell, in which case the selective depletion complex may be purified from the cell culture media. The selective depletion complex may be captured by chromatography, such as by a protein A column or a Ni-affinity column, through use of any manner of expressed affinity tags, size or ion exchange chromatography, and then purified by one or more steps, which may include chromatography, and then optionally buffer exchanged. The selective depletion complexes of this disclosure may be advantageously manufactured by standard manufacturing methods for recombinant proteins or recombinant Fc- containing molecules, such as those described in Shukla et a., 2017 Bioengineering & Translational Medicine 2017: 2:58-69.

[0412] In the case of cell- or viral-based samples, organisms can be treated prior to purification to preserve and/or release a target polypeptide. In some embodiments, the cells are fixed using a fixing agent. In some embodiments, the cells are lysed. The cellular material can be treated in a manner that does not disrupt a significant proportion of cells, but which removes proteins from the surface of the cellular material, and/or from the interstices between cells. For example, cellular material can be soaked in a liquid buffer, or, in the case of plant material, can be subjected to a vacuum, in order to remove proteins located in the intercellular spaces and/or in the plant cell wall. If the cellular material is a microorganism, proteins can be extracted from the microorganism culture medium. Alternatively, the peptides can be packed in inclusion bodies. The inclusion bodies can further be separated from the cellular components in the medium. In some embodiments, the cells are not disrupted. A cellular or viral peptide that is presented by a cell or virus can be used for the attachment and/or purification of intact cells or viral particles. In addition to recombinant systems, peptides can also be synthesized in a cell-free system prior to extraction using a variety of known techniques employed in protein and peptide synthesis.

[0413] In some cases, a host cell produces a peptide that has an attachment point for a cargo molecule (e.g., a therapeutic agent). An attachment point could comprise a lysine residue, an N- terminus, a cysteine residue, a cysteine disulfide bond, a glutamic acid or aspartic acid residue, a C-terminus, or a non-natural amino acid. The peptide could also be produced synthetically, such as by solid-phase peptide synthesis, or solution-phase peptide synthesis. Peptide synthesis can be performed by fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. The peptide could be folded (formation of disulfide bonds) during synthesis or after synthesis or both. Peptide fragments could be produced synthetically or recombinantly. Peptide fragments can be then be joined together enzymatically or synthetically.

[0414] In other aspects, the peptides of the present disclosure can be prepared by conventional solid phase chemical synthesis techniques, for example according to the Fmoc solid phase peptide synthesis method (“Fmoc solid phase peptide synthesis, a practical approach,” edited by W. C. Chan and P. D. White, Oxford University Press, 2000).

[0415] In some embodiments, the peptides of this disclosure can be more stable during manufacturing. For example, peptides of this disclosure can be more stable during recombinant expression and purification, resulting in lower rates of degradation by proteases that are present in the manufacturing process, a higher purity of peptide, a higher yield of peptide, or any combination thereof. In some embodiments, the peptides can also be more stable to degradation at high temperatures and low temperatures during manufacturing, storage, and distribution. For example, in some embodiments peptides of this disclosure can be stable at 25 °C. In other embodiments, peptides of this disclosure can be stable at 70 °C or higher than 70 °C. In some embodiments, peptides of this disclosure can be stable at 100 °C or higher than 100 °C.

Pharmaceutical Compositions

[0416] A pharmaceutical composition of the disclosure can be a combination of any peptide as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, antioxidants, solubilizers, buffers, osmolytes, salts, surfactants, amino acids, encapsulating agents, bulking agents, cryoprotectants, and/or excipients. The pharmaceutical composition facilitates administration of a peptide described herein to an organism. In some cases, the pharmaceutical composition comprises factors that extend half-life of the peptide and/or help the peptide to penetrate the target cells. In some embodiments, a pharmaceutical composition comprises a cell modified to express and secrete a selective depletion complex of the present disclosure.

[0417] Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intratumoral, intranasal, and topical administration. A pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the peptide described herein directly into an organ, optionally in a depot.

[0418] Parenteral injections can be formulated for bolus injection, infusion, or continuous infusion. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of a peptide described herein in water-soluble form. Suspensions of peptide-antibody complexes described herein can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduce the aggregation of such peptide-antibody complexes described herein to allow for the preparation of highly concentrated solutions.

[0419] Alternatively, the peptide described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, water for injection, or a formulated buffer before use. In some embodiments, a purified peptide is administered intravenously. A peptide described herein can be administered to a subject in order to home, target, migrate to, or be directed to a CNS cell, a brain cell, a cancerous cell, or a tumor. In some embodiments, a peptide can be conjugated to, linked to, or fused to another peptide that provides a targeting function to a specific target cell type in the central nervous system or across the blood brain barrier. Exemplary target cells include a CNS cell, erythrocyte, an erythrocyte precursor cell, an immune cell, a stem cell, a muscle cell, a brain cell, a thyroid cell, a parathyroid cell, an adrenal gland cell, a bone marrow cell, an appendix cell, a lymph node cell, a tonsil cell, a spleen cell, a muscle cell, a liver cell, a gallbladder cell, a pancreas cell, a cell of the gastrointestinal tract, a glandular cell, a kidney cell, a urinary bladder cell, an endothelial cell, an epithelial cell, a choroid plexus epithelial cell, a neuron, a glial cell, an astrocyte, or a cell associated with a nervous system.

[0420] A peptide of the disclosure can be applied directly to an organ, or an organ tissue or cells, such as brain or brain tissue or cells, during a surgical procedure. The recombinant peptide described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

[0421] In practicing the methods of treatment or use provided herein, therapeutically effective amounts of a peptide described herein can be administered in pharmaceutical compositions to a subject suffering from a condition that affects the immune system. In some embodiments, the subject is a mammal such as a human or a primate. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

[0422] In some embodiments, a peptide is cloned into a viral or non-viral expression vector. Such expression vector can be packaged in a viral particle, a virion, or a non-viral carrier or delivery mechanism, which is administered to patients in the form of gene therapy. In other embodiments, patient cells are extracted and modified to express a peptide capable of binding TfR ex vivo before the modified cells are returned back to the patient in the form of a cell-based therapy, such that the modified cells will express the peptide once transplanted back in the patient.

[0423] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a peptide described herein can be manufactured, for example, by expressing the peptide in a recombinant system, purifying the peptide, lyophilizing the peptide, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes. The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form.

[0424] Methods for the preparation of peptide described herein comprising the compounds described herein include formulating peptide described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

[0425] Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington ’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0426] Pharmaceutical compositions can also include permeation or absorption enhancers (Aungst et al. AAPS J. 14(1): 10-8. (2012) and Moroz et al. Adv Drug Deliv Rev 101:108-21.

(2016)). Permeation enhancers can facilitate uptake of molecules from the GI tract into systemic circulation. Permeation enhancers can include salts of medium chain fatty acids, sodium caprate, sodium caprylate, N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), N-(5- chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), hydrophilic aromatic alcohols such as phenoxyethanol, benzyl alcohol, and phenyl alcohol, chitosan, alkyl glycosides, dodecyl-2-N,N- dimethylamino propionate (DDAIPP), chelators of divalent cations including EDTA, EGTA, and citric acid, sodium alkyl sulfate, sodium salicylate, lecithin-based, or bile salt-derived agents such as deoxy cholates.

[0427] Compositions can also include protease inhibitors including soybean trypsin inhibitor, aprotinin, sodium glycocholate, camostat mesilate, vacitracin, or cyclopentadecalactone. Use of Peptides in Treatments

[0428] In some embodiments, a method of treating a subject using the selective depletion complexes of the present disclosure includes administering an effective amount of a peptide as described herein to a subject in need thereof.

[0429] In some embodiments, a method of treating a subject using the selective depletion complexes of the present disclosure includes modifying a cell of a subject to express and secrete a selective depletion complex of the present disclosure. In some embodiments, the cell is a cell in the subject. In some embodiments, the cell is a cell that has been removed from the subject and is re-introduced following modification. In some embodiments, the cell is modified using a viral vector (e.g., an oncolytic herpes simplex virus). In some embodiments, a gene encoding expression and secretion of a selective depletion complex is engineered into a CAR-T cell or other cellular therapy.

[0430] TfR can be expressed in various tissues such as the brain, the stomach, the liver, of the gall bladder. Hence, the peptides of the present disclosure (e.g., a selective depletion complex comprising a TfR-b inding peptide) can be used in the diagnosis and treatment of disease and conditions associated with various tissues and organs. For example, drug delivery to these tissues and organs can be improved by using the herein described peptides and peptide complexes carrying a diagnostic and/or therapeutic payload.

[0431] The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case can be determined using techniques, such as a dose escalation study.

[0432] The methods, compositions, and kits of this disclosure can comprise a method to prevent, treat, arrest, reverse, or ameliorate the symptoms of a condition. The treatment can comprise treating a subject (e.g., an individual, a domestic animal, a wild animal, or a lab animal afflicted with a disease or condition) with a peptide of the disclosure. The disease can be a cancer or tumor. In treating the disease, the peptide can contact the tumor or cancerous cells. The subject can be a human. Subjects can be humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, and fetuses in utero. [0433] Treatment can be provided to the subject before clinical onset of disease. Treatment can be provided to the subject after clinical onset of disease. Treatment can be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of the disease. Treatment can be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment can be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment can also include treating a human in a clinical trial. A treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure. A treatment can comprise a once daily dosing. A treatment can comprise delivering a peptide of the disclosure to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, dermally, topically, by intra-articular injection, orally, sublingually, intrathecally, transdermally, intranasally, via a peritoneal route, directly into a tumor e.g., injection directly into a tumor, directly into the brain, e.g., via and intracerebral ventricle route, or directly onto a joint, e.g. via topical, intra-articular injection route. A treatment can comprise administering a peptide-active agent complex to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, by intra-articular injection, dermally, topically, orally, intrathecally, transdermally, intransally, parenterally, orally, via a peritoneal route, nasally, sublingually, or directly onto cancerous tissues.

[0434] In some embodiments, a target-binding peptide (e.g., a target-binding EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390, SEQ ID NO: 457 - SEQ ID NO: 494) may be administered for a therapeutic effect. For example, the target-binding peptide may bind to and inhibit an EGFR receptor, producing a therapeutic effect in a subject. In some embodiments, the target-binding peptide may be complexed with an active agent (e.g., a therapeutic agent or a detectable agent). For example, an EGFR-b inding peptide may be complexed with an anticancer agent (e.g., a chemotherapeutic agent). Administration of an EGFR-binding peptide complexed with an anti-cancer agent may be used in a method of treating a disease or condition (e.g., cancer).

Peptide Kits

[0435] In one aspect, peptides described herein can be provided as a kit. In another embodiment, peptide complexes described herein can be provided as a kit. In another embodiment, a kit comprises amino acids encoding a peptide described herein, a vector, a host organism, and an instruction manual. In some embodiments, a kit includes written instructions on the use or administration of the peptides.

[0436] Additional aspects and advantages of the present disclosure will become apparent to those skilled in this art from the following detailed description, wherein illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Numbered Embodiments

[0437] The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. An EGFR-b inding peptide comprising a sequence having at least 70% sequence identity to SEQ ID NO: 317 and comprising at least one mutation relative to SEQ ID NO: 317. 2. The EGFR- binding peptide of embodiment 1 , comprising a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue; wherein the EGFR-b inding peptide comprises: seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue. 3. The EGFR-binding peptide of embodiment 2, wherein: the first cysteine amino acid residue is at position 6 of the EGFR-binding peptide, the second cysteine amino acid residue is at position 14 of the EGFR-binding peptide, the third cysteine amino acid residue is at position 20 of the EGFR-binding peptide, the fourth cysteine amino acid residue is at position 31 of the EGFR-binding peptide, the fifth cysteine amino acid residue is at position 33 of the EGFR-binding peptide, and the sixth cysteine amino acid residue is at position 42 of the EGFR-binding peptide. 4. The EGFR-binding peptide of any one of embodiments 1-3, wherein the at least one mutation comprises an amino acid substitution of D11R, I23S, V35E, S51P, L52E, R53E, M21R, A30W, I38D, W49R, V34S, Q43I, Q43V, Q43W, Q43Y, K48N, K48T, K48A, K48L, E51S, E51H, L52H, R53H, or a combination thereof. 5. The EGFR-binding peptide of any one of embodiments 1-4, wherein the at least one mutation comprises an amino acid substitution of M21R, A30W, I38D, W49R, or a combination thereof. 6. The EGFR-binding peptide of any one of embodiments 1-5, wherein the at least one mutation comprises an amino acid substitution of DI 1R, I23S, V35E, S51P, L52E, R53E, or a combination thereof. 7. The EGFR-binding peptide of any one of embodiments 1-6, wherein the at least one mutation comprises an amino acid substitution of E51H, L52H, R53H, or a combination thereof. 8. An EGFR-binding peptide comprising a sequence of SEQ ID NO: 314. 9. An EGFR-binding peptide comprising a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. 10. The EGFR-binding peptide of embodiment 9, comprising a sequence having at least 90% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. 11. The EGFR-binding peptide of any one of embodiments 1-10, wherein the EGFR-binding peptide is capable of binding to EGFR without activating the EGFR. 12. The EGFR-binding peptide of any one of embodiments 1-11, wherein the EGFR-binding peptide blocks binding of EGF to EGFR when the EGFR-binding peptide is bound to the EGFR. 13. The EGFR-binding peptide of any one of embodiments 1-12, wherein the EGFR-binding peptide inhibits EGFR when the EGFR-binding peptide is bound to the EGFR. 14. The EGFR-binding peptide of any one of embodiments 1-13, wherein the EGFR- binding peptide prevents dimerization of EGFR when the EGFR-binding peptide is bound to the EGFR. 15. A peptide complex comprising: (a) a cellular receptor-binding peptide; and (b) a target-binding peptide complexed with the cellular receptor-binding peptide, wherein the targetbinding peptide has affinity for a target molecule, and wherein the target-binding peptide comprises the EGFR-binding peptide of any one of embodiments 1-14. 16. A peptide complex comprising: (a) a cellular receptor-binding peptide; and (b) a target-binding peptide complexed with the cellular receptor-binding peptide, wherein the target-binding peptide has affinity for a target molecule, and wherein the target-binding peptide comprises a sequence of SEQ ID NO: 314 or a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. 17. The peptide complex of embodiment 15 or embodiment 16, wherein the affinity of the target-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH-independent. 18. The peptide complex of embodiment 15 or embodiment 16, wherein the affinity of the target-binding peptide for the target molecule, the affinity of the cellular receptor binding peptide for the cellular receptor, or both is pH dependent. 19. The peptide complex of any one of embodiments 15-18, wherein the affinity of the targetbinding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for the cellular receptor, or both is ionic strength dependent. 20. The peptide complex of any one of embodiments 15-19, wherein the target binding peptide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. 21. The peptide complex of any one of embodiments 15-20, wherein the target binding peptide comprises a sequence of any one of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. 22. The peptide complex of any one of embodiments 15-21, wherein the cellular receptor-binding peptide is a transferrin receptorbinding peptide or a PD-L1 -binding peptide. 23. The peptide complex of any one of embodiments 15-22, wherein the cellular receptor is a transferrin receptor or PD-L1. 24. The peptide complex of any one of embodiments 15-23, wherein the cellular receptor is a cationindependent mannose 6 phosphate receptor (CI-M6PR), an asialoglycoprotein receptor (ASGPR), CXCR7, folate receptor, or Fc receptor (including but not limited to neonatal Fc receptor (FcRn) or FcyRIIb). 25. The peptide complex of any one of embodiments 15-24, wherein the cellular receptor-binding peptide binds to the cellular receptor at a pH of from pH 4.5 to pH 7.4, from pH 5.5 to pH 7.4, from pH 5.8 to pH 7.4, or from pH 6.5 to pH 7.4. 26. The peptide complex of any one of embodiments 15-25, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4. 27. The peptide complex of any one of embodiments 15-26, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5. 28. The peptide complex of any one of embodiments 15-27, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with a dissociation rate constant (koff or kd) of no more than 1 s'¹, no more than 5x1 O'¹ s'¹, no more than 2x1 O'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than IxlO'³ s'¹, no more than 5xl0'⁴ s'¹, or no more than 2xl0'⁴ s'¹ at pH 5.5. 29. The peptide complex of any one of embodiments 15-28, wherein the affinity of the cellular receptorbinding peptide for the cellular receptor is pH-independent. 30. The peptide complex of any one of embodiments 15-29, wherein the affinity of the target-binding peptide for the target molecule is pH-dependent. 31. The peptide complex of any one of embodiments 15-29, wherein the affinity of the target-binding peptide for the target molecule is pH-independent. 32. The peptide complex of any one of embodiments 15-31, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. 33. The peptide complex of any one of embodiments 15-32, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. 34. The peptide complex of any one of embodiments 15-32, wherein the dissociation rate constant (koff or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. 35. The peptide complex of any one of embodiments 15-32, wherein the dissociation rate constant (koff or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5 -fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. 36. The peptide complex of any one of embodiments 15- 35, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor is pH dependent. 37. The peptide complex of embodiment 36, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor decreases as pH decreases. 38. The peptide complex of embodiment 36 or embodiment 37, wherein the affinity of the cellular receptorbinding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.5. 39. The peptide complex of embodiment 36 or embodiment 37, wherein the affinity of the cellular receptorbinding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.8. 40. The peptide complex of any one of embodiments 15-39, wherein the affinity of the target-binding peptide for the target molecule is pH dependent. 41. The peptide complex of any one of embodiments 15- 40, wherein the affinity of the target-binding peptide for the target molecule decreases as pH decreases. 42. The peptide complex of any one of embodiments 15-41, wherein the affinity of the target-binding peptide for the target molecule is higher at a higher pH than at a lower pH. 43. The peptide complex of embodiment 42, wherein the higher pH is pH 7.4, pH 7.2, pH 7.0, or pH 6.8. 44. The peptide complex of embodiment 42 or embodiment 43, wherein the lower pH is pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. 45. The peptide complex of any one of embodiments 15-44, wherein the affinity of the target-binding peptide for the target molecule is higher at pH 7.4 than at pH 6.0. 46. The peptide complex of any one of embodiments 15-45, wherein the affinity of the target-binding peptide for the target molecule is higher at pH 7.4 than at pH 5.5. 47. The peptide complex of any one of embodiments 15-46, wherein the affinity of the target-binding peptide for the target molecule is higher at pH 7.4 than at pH 5.8. 48. The peptide complex of any one of embodiments 15-47, wherein the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no more than 500 nM, no more than 200 nM, 100 nM, no more than 50 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 1 nM, or no more than 0.1 nM at pH 7.4. 49. The peptide complex of any one of embodiments 15-48, wherein the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1x10'¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x1 O’⁴ s’¹, no more than 1x1 O’⁴ s’¹, no more than 5x10’⁵ s’¹, or no more than 2xl0’⁵ s’¹ at pH 7.4. 50. The peptide complex of any one of embodiments 15-49, wherein the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’ no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, or no more than 2xl0’⁴ s’¹ at pH 5.5. 51. The peptide complex of any one of embodiments 15-50, wherein the target-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s’¹, no more than 5x10’¹ s’¹, no more than 2x10’¹ s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5xl0'⁴ s'¹, or no more than 2xl0'⁴ s'¹ at pH 5.8. 52. The peptide complex of any one of embodiments 15-51, wherein the dissociation rate constant (koff or kd) for target-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.5 than at pH 7.4. 53. The peptide complex of any one of embodiments 15-52, wherein the dissociation rate constant (koff or kd) for target-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.8 than at pH 7.4. 54. The peptide complex of any one of embodiments 15-53, wherein the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.5. 55. The peptide complex of any one of embodiments 15-54, wherein the target-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.8. 56. The peptide complex of any one of embodiments 15-55, wherein the affinity of the target-binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the targetbinding peptide for the target molecule at pH 5.5. 57. The peptide complex of any one of embodiments 15-56, wherein the affinity of the target-binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20- fold greater than the affinity of the target-binding peptide for the target molecule at pH 5.8. 58. The peptide complex of any one of embodiments 15-57, wherein the affinity of the targetbinding peptide for the target molecule at pH 7.4 is less than 0.5-fold, less than 1-fold, less than, 1.5-fold, less than 2-fold, less than 3-fold, or less than 10-fold, greater than the affinity of the target-binding peptide for the target molecule at pH 5.8. 59. The peptide complex of any one of embodiments 15-58, wherein the target-binding peptide comprises one or more histidine amino acid residues. 60. The peptide complex of any one of embodiments 15-59, wherein the affinity of the target-binding peptide for the target molecule decreases as ionic strength increases. 61. The peptide complex of any one of embodiments 15-60, wherein the target-binding peptide comprises one or more polar or charged amino acid residues capable of forming polar or chargecharge interactions with the target molecule. 62. The peptide complex of any one of embodiments 15-61, wherein the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the target-binding peptide. 63. The peptide complex of any one of embodiments 15-62, wherein the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the target-binding peptide via a polymer linker. 64. The peptide complex of embodiment 63 wherein the polymer linker is a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer comprising proline, alanine, serine, or a combination thereof, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, a palmitic acid, an albumin, or an albumin binding molecule.

65. The peptide complex of any one of embodiments 15-64, wherein the cellular receptorbinding peptide and the target-binding peptide form a single polypeptide chain. 66. The peptide complex of any one of embodiments 15-65, wherein the peptide complex comprises a dimer dimerized via a dimerization domain. 67. The peptide complex of any one of embodiments 62-

66, wherein the distance between the cellular receptor-binding peptide and the target-binding peptide is at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, or at least 100 nm. 68. The peptide complex of embodiment 66, wherein the dimerization domain comprises an Fc domain. 69. The peptide complex of embodiment 66, wherein the dimer is a homodimer dimerized via a homodimerization domain. 70. The peptide complex of embodiment 69, wherein the homodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 535, SEQ ID NO: 706, or SEQ ID NO: 246. 71. The peptide complex of embodiment 66, wherein the dimer is a heterodimer dimerized via a first heterodimerization domain and a second heterodimerization domain. 72. The peptide complex of embodiment 71, wherein the first heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 536, SEQ ID NO: 707, or SEQ ID NO: 709. 73. The peptide complex of embodiment 71 or embodiment 72, wherein the second heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 537, SEQ ID NO: 708, or SEQ ID NO: 710. 74. The peptide complex of any one of embodiments 66-73, wherein the target-binding peptide is linked to the dimerization domain via a peptide linker. 75. The peptide complex of any one of embodiments 66-74, wherein the cellular receptor-binding peptide is linked to the dimerization domain via a peptide linker. 76. The peptide complex of any one of embodiments 15-75, wherein the cellular receptor-binding peptide is linked to the target-binding peptide via a peptide linker. 77. The peptide complex of embodiment 15-76, wherein the peptide linker has a length of from 1 to 50 amino acid residues, from 2 to 40 amino acid residues, from 3 to 20 amino acid residues, or from 3 to 10 amino acid residues. 78. The peptide complex of any one of embodiments 75-77, wherein the peptide linker comprises glycine and serine amino acids. 79. The peptide complex of any one of embodiments 75-78, wherein the peptide linker has a persistence length of no more than 6 A, no more than 8 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, no more than 50 A, no more than 75 A, no more than 100 A, no more than 150 A, no more than 200 A, no more than 250 A, or no more than 300 A. 80. The peptide complex of any one of embodiments 75-79, wherein the peptide linker is derived from an immunoglobulin peptide. 81. The peptide complex of any one of embodiments 75-80, wherein the peptide linker is derived from a double-knot toxin peptide. 82. The peptide complex of any one of embodiments 75-81, wherein the peptide linker comprises a sequence of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 223 - SEQ ID NO: 223 - SEQ ID NO: 227, SEQ ID NO: 194, SEQ ID NO: 391, SEQ ID NO: 538, or SEQ ID NO: 540 - SEQ ID NO: 541. 83. The peptide complex of any one of embodiments 15-82, wherein the cellular receptorbinding peptide, the target-binding peptide, peptide complex, or a combination thereof comprises a miniprotein, a nanobody, an antibody, an antibody fragment, an scFv, a DARPin, or an affibody. 84. The peptide complex of embodiment 83, wherein the antibody comprises an IgG, or wherein the antibody fragment comprises a Fab, a F(ab)2, an scFv, or an (scFv)2. 85.

The peptide complex of embodiment 83 or embodiment 84, wherein the miniprotein comprises a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide. 86. The peptide complex of any one of embodiments 15-85, wherein the cellular receptor-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. 87. The peptide complex of any one of embodiments 15-86, wherein the targetbinding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. 88. The peptide complex of any one of embodiments 15-86, wherein the peptide complex comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds. 89. The peptide complex of any one of embodiments 15-88, wherein the cellular receptor-binding peptide comprises at least six cysteine residues. 90. The peptide complex of embodiment 89, wherein the at least six cysteine residues are positioned at amino acid positions 4, 8, 18, 32, 42, and 46 of the cellular receptor-binding peptide. 91. The peptide complex of embodiment 89 or embodiment 90, wherein the at least six cysteine residues form at least three disulfide bonds. 92. The peptide complex of any one of embodiments 15-91, wherein the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 148 - SEQ ID NO: 177. 93. The peptide complex of any one of embodiments 15-92, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64. 94. The peptide complex of any one of embodiments 15-93, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 96. 95. The peptide complex of any one of embodiments 15-94, wherein the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 96. 96. The peptide complex of any one of embodiments 15-91, wherein the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 392 - SEQ ID NO: 399. 97. The peptide complex of any one of embodiments 15-91 or embodiment 96, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241. 98. The peptide complex of embodiment 96 or embodiment 97, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 187, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 400, or SEQ ID NO: 401 or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 187. 99. The peptide complex of any one of embodiments 96-98, wherein the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 187. 100. The peptide complex of any one of embodiments 96-99, wherein the fragment comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues. 101. The peptide complex of any one of embodiments 15-100, wherein the cellular receptor-binding peptide comprises one or more histidine residues at a cellular receptor-binding interface. 102. The peptide complex of any one of embodiments 15-101, wherein the target-binding peptide comprises one or more histidine residues at a target-binding interface. 103. The peptide complex of any one of embodiments 15- 102, wherein the target-binding peptide is an EGFR-binding peptide. 104. The peptide complex of any one of embodiments 15-103, wherein the target molecule comprises an EGFR. 105. The peptide complex of embodiment 104, wherein the EGFR is wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, or a combination thereof. 106. The peptide complex of embodiment 105, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant. 107. The peptide complex of any one of embodiments 15-106, wherein an off rate of the cellular receptor-binding peptide from the cellular receptor is slower than a recycling rate of the cellular receptor. 108. The peptide complex of any one of embodiments 15-107, wherein a half-life of dissociation of the cellular receptor-binding peptide from the cellular receptor is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, no faster than 20 minutes, no faster than 30 minutes, no faster than 45 minutes, no faster than 60 minutes, no faster than 90 minutes, or no faster than 120 minutes. 109. The peptide complex of any one of embodiments 15-108, wherein a rate of dissociation of the target-binding peptide from the target molecule is faster than a recycling rate of the cellular receptor. 110. The peptide complex of any one of embodiments 15-109, wherein a half-life of dissociation of the target binding-binding peptide from the target molecule is less than 10 seconds, less than 20 seconds, less than 30 seconds, less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, or less than 60 minutes in endosomal conditions. 111. The peptide complex of any one of embodiments 15-110, wherein the peptide complex is capable of being endocytosed via receptor-mediated endocytosis. 112. The peptide complex of embodiment 111, wherein the receptor-mediated endocytosis is transferrin receptor- mediated endocytosis. 113. The peptide complex of embodiment 112, wherein the receptor- mediated endocytosis is PD-L1 -mediated endocytosis. 114. The peptide complex of any one of embodiments 15-113, wherein the cellular receptor-binding peptide remains bound to the cellular receptor inside an endocytic vesicle. 115. The peptide complex of any one of embodiments 15-114, wherein the peptide complex is recycled to the cell surface when the cellular receptor-binding peptide is bound to the cellular receptor and the cellular receptor is recycled. 116. The peptide complex of any one of embodiments 15-115, wherein the target molecule is released or dissociated from the target-binding peptide after the peptide complex is endocytosed via receptor-mediated endocytosis. 117. The peptide complex of any one of embodiments 15-116, wherein the target molecule is an extracellular protein, a circulating protein, or a soluble protein. 118. The peptide complex of any one of embodiments 15-117, wherein the target molecule is a cell surface protein. 119. The peptide complex of any one of embodiments 15-118, wherein the target molecule is a transmembrane protein. 120. The peptide complex of any one of embodiments 15-119, further comprising a half-life modifying agent coupled to the cellular receptor-binding peptide, the target-binding peptide, or both. 121. The peptide complex of embodiment 120, wherein the half-life modifying agent is a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin. 122. The peptide complex of embodiment 121, wherein the molecule that binds to albumin is a serum albumin-binding peptide. 123. The peptide complex of embodiment 122, wherein the serum albumin-binding peptide comprises a sequence of any one of SEQ ID NO: 178, SEQ ID NO: 179, or SEQ ID NO: 193. 124. The peptide complex of any one of embodiments 1-123, wherein the cellular receptor-binding peptide, the target-binding peptide, or both is recombinantly expressed. 125. The peptide complex of any one of embodiments 1-124, wherein the target-binding peptide is configured to dissociate from the target molecule at pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5. 126. The peptide complex of any one of embodiments 1-125, wherein the cellular receptor-binding peptide is configured to dissociate from the cellular receptor at pH 6.5, pH 6.0, pH 5.5, pH 5.0, or pH 4.5. 127. The peptide complex of any one of embodiments 1-126, wherein the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 507, SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 516. 128. The peptide complex of any one of embodiments 1-126, wherein the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 503, or SEQ ID NO: 506. 129. A peptide-active agent complex comprising a peptide complexed to an active agent, wherein the peptide comprises the EGFR-binding peptide of any one of embodiments 1-14 or the peptide complex of any one of embodiments 15-128. 130. The peptideactive agent complex of embodiment 129, wherein the active agent comprises a peptide, a peptidomimetic, an oligonucleotide, a DNA, an RNA, an antibody, a single chain variable fragment (scFv), an antibody fragment, an aptamer, or a small molecule. 131. The peptide-active agent complex of embodiment 130, wherein the DNA comprises cDNA, ssDNA, or dsDNA. 132. The peptide-active agent complex of embodiment 130, wherein the RNA comprises RNAi, microRNA, snRNA, dsRNA, or an antisense oligonucleotide. 133. The peptide-active agent complex of embodiment 129, wherein the active agent is a therapeutic agent or a detectable agent. 134. The peptide-active agent complex of embodiment 133, wherein the detectable agent comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemi luminescent compound, a radioisotope, nanoparticle, a paramagnetic metal ion, or a combination thereof. 135. The peptide-active agent complex of embodiment 133, wherein the therapeutic agent is an anti-cancer agent. 136. The peptide-active agent complex of embodiment 135, wherein the anti-cancer agent comprises a radionuclide, radioisotope, a chemotherapeutic agent, a platinum therapeutic, a toxin, an enzyme, a sensitizing drug, an anti-angiogenic agent, cisplatin, an anti-metabolite, an anti-metabolic therapeutic, a mitotic inhibitor, a growth factor inhibitor, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, or amifostine. 137. The peptide-active agent complex of embodiment 135 or embodiment 136, wherein the anti-cancer agent targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. 138. A pharmaceutical composition comprising the EGFR- binding peptide of any one of embodiments 1-14 and a pharmaceutically acceptable excipient or diluent. 139. A pharmaceutical composition comprising the peptide complex of any one of embodiments 15-128 or the peptide-active agent complex of any one of embodiments 129-137 and a pharmaceutically acceptable excipient or diluent. 140. A method of inhibiting EGFR in a subject, the method comprising administering to the subject a composition comprising the EGFR-b inding peptide of any one of embodiments 1-14 and delivering the EGFR-b inding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-b inding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR. 141. The method of embodiment 140, wherein the EGFR-binding peptide inhibits activation of the EGFR by disrupting multimerization, dimerization, or heterodimerization of the EGFR on the cell of the subject that expresses EGFR. 142. A method of selectively depleting a target molecule, the method comprising: (a) contacting the peptide complex of any one of embodiments 15-128 to a cell expressing a cellular receptor; (b) binding the target-binding peptide to the target molecule under extracellular conditions; (c) binding the cellular receptor-binding peptide to the cellular receptor under extracellular conditions; and (d) endocytosing the peptide complex, the target molecule, and the cellular receptor into an endocytic or lysosomal compartment, thereby depleting the target molecule. 143. The method of embodiment 142, further comprising: (e) dissociating the target-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both under endosomal or lysosomal conditions. 144. The method of embodiment 142 or embodiment 143, further comprising: (f) degrading the target molecule, thereby further depleting the target molecule. 145. The method of any one of embodiments 142-144, further comprising recycling the peptide complex and the cellular receptor to the cell surface. 146. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the EGFR-b inding peptide of any one of embodiments 1-14 or the pharmaceutical composition of embodiment 138 and delivering the EGFR-b inding peptide to a cell of the subject that expresses EGFR, wherein the EGFR- binding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition. 147. The method of embodiment 146, wherein the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, or EGFR mutant T790M. 148. The method of embodiment 147, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant. 149. A method of treating a disease or condition in a subject in need thereof, the method comprising: (a) administering to the subject the EGFR-binding peptide of any one of embodiments 1-14, the peptide complex of any one of embodiments 15-128, the peptide-active agent complex of any one of embodiments 129-137, or the pharmaceutical composition of embodiment 138 or embodiment 139; (b) binding the targetbinding peptide under extracellular conditions to a target molecule associated with the disease or condition on a cell of the subject expressing the target molecule and a cellular receptor; (c) binding the cellular receptor-binding peptide under extracellular conditions to the cellular receptor on the cell of the subject; and (d) endocytosing the peptide complex, the target molecule, and the cellular receptor. 150. The method of embodiment 149, further comprising: (e) dissociating the target-binding peptide from the target molecule, the cellular-receptor-binding peptide from the cellular receptor, or both under endosomal conditions. 151. The method of any one of embodiments 149 or embodiment 150, further comprising: (f) degrading the target molecule. 152. The method of any one of embodiments 149-151, wherein the target molecule comprises an EGFR. 153. The method of embodiment 152, wherein the EGFR comprises wildtype EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, or EGFR mutant T790M. 154. The method of embodiment 153, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant. 155. The method of any one of embodiments 149-154, wherein the disease or condition is a cancer. 156. The method of embodiment 155, wherein the cancer expresses EGFR, overexpresses EGFR, or contains mutant EGFR. 157. The method of embodiment 155 or embodiment 156, wherein the cancer is breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, blood-cell-derived cancer, lung cancer, sarcoma, stomach cancer, a gastrointestinal cancer, glioblastoma, head and neck cancer, squamous head and neck cancer, non-small-cell lung cancer, squamous non-small cell lung cancer, pancreatic cancer, ovarian cancer, endometrial cancer, blood cancer, skin cancer, liver cancer, kidney cancer, or colorectal cancer. 158. The method of any one of embodiments 155-157, wherein the cancer is TKI-resistant, cetuximab-resistant, necitumumab-resistant, or panitumumab-resistant. 159. The method of any one of embodiments 155-158, wherein the cancer has one or more of the following: overexpresses EGFR, KRAS mutation, KRAS G12S mutation, KRAS G12C mutation, PTEN loss, EGFR exonl9 deletion, EGFR L858R mutation, EGFR T790M mutation, a cetuximabresistant EGFR, a panitumumab-resistant EGFR, PIK3CA mutation, TP53 R273H mutation, PIK3CA amplification, PIK3CA G118D, TP53 R273H, EGFR C797X mutation, EGFR G724S mutation, EGFR L718Q mutation, EGFR S768I mutation, an EGFR mutation, or a combination thereof. 160. The method of embodiment any one of embodiments 155-159, wherein the cancer expresses or has upregulated c-MET, Her2, Her3 that heterodimerizes with EGFR. 161. The method of any one of embodiments 155-160, wherein the cancer is a primary cancer, an advanced cancer, a metastatic cancer, a metastatic cancer in the central nervous system, a primary cancer in the central nervous system, metastatic colorectal cancer, metastatic head and neck cancer, metastatic non-small-cell lung cancer, metastatic breast cancer, metastatic skin cancer, a refractory cancer, a KRAS wild type cancer, a KRAS mutant cancer, or an exon20 mutant non-small-cell lung cancer. 162. The method of any one of embodiments 149-161, further comprising administering an additional therapy to the subject. 163. The method of embodiment 162, where the additional therapy is adjuvant, first-line, or combination therapy. 164. The method of embodiment 162 or embodiment 163, wherein the additional therapy targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells. 165. The method of embodiment 164, wherein targeting of other oncogenic signaling pathways comprises administration of inhibitors of MEK/ERK pathway signaling, PI3K/AKT pathway signaling, JAK/STAT pathway signaling, or WNT/p- catenin pathway signaling. 166. The method of embodiment 164, wherein targeting of immune response pathways comprises PD-1/PD-L1 checkpoint inhibition. 167. The method of embodiment 164, wherein directly driving an immune response to cancer cells comprises bispecific T cell engagers or chimeric antigen receptor expressing T cells. 168. The method of embodiment 164, wherein the targeting disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells comprises administering seno lytic agents to a subject. 169. The method of any one of embodiments 162-168, wherein the additional therapy comprises administering fluorouracil, FOLFIRI, irinotecan, FOLFOX, gemcitabine, or cisplatin, irinotecan, oxiplatin, fluoropyrimidine to the subject. 170. The method of any one of embodiments 142-145 or 149-169, further comprising forming a ternary complex between the selective depletion complex, the target molecule, and the cellular receptor. 171. The method of embodiment 170, wherein formation of the ternary complex increases, facilitates, or stabilizes recycling or turnover of the cellular receptor, the target molecule, or both. 172. The method of embodiment 170 or embodiment 171, wherein formation of the ternary complex increases, facilitates, or stabilizes binding of the target molecule to the cellular receptor. 173. The method of embodiment 142-145 or 149-172, wherein the peptide complex binds at higher levels to cells that overexpress the target molecule and the cellular receptor than to cells that have lower levels of the target molecule or the cellular receptor or both. 174. The method of any one of embodiments 142-145 or 149-173, wherein the peptide complex has a larger, longer, or wider therapeutic window as compared to an alternative therapy. 175. The method of embodiment 174, wherein the alternative therapy is not recycled to the cell surface. 176. The method of embodiment 174 or embodiment 175, wherein the alternative therapy is a lysosomal targeting therapy, a ubiquitin-proteosome system (UPS) targeting therapy, a non-selective therapeutic agent, an existing biologic, or a lysosomal delivery molecule. 177. The method of any one of embodiments 142-176, wherein the peptide complex or the EGFR-binding peptide is administered at lower molar dosage than alternative therapies. 178. The method of embodiment 142-177, wherein the peptide complex or the EGFR-binding peptide binds at higher levels to cancer cells than to normal cells. 179. The method of embodiment 142-178, wherein the peptide complex or the EGFR-binding peptide has a higher antiproliferative effect, a higher target molecule depletion effect, or a higher viability effect on cancer cells than on normal cells in vitro or in vivo. 180. The method of embodiment 142-179, wherein the peptide complex or the EGFR-binding peptide has a larger, longer, or wider therapeutic window than an anti-EGFR antibody or a TKI. 181. The method of embodiment 142-180, wherein the peptide complex or the EGFR-binding peptide has lower toxicity on skin or on keratinocytes than an anti-EGFR antibody or a TKI. 182. A method of administering a peptide complex or an EGFR-binding peptide to a subject, the method comprising administering the EGFR-binding peptide of any one of embodiments 1-14, the peptide complex of any one of embodiments 15-128, the peptideactive agent complex of any one of embodiments 129-137, or the pharmaceutical composition of embodiment 138 or embodiment 139. 183. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the EGFR-binding peptide of any one of embodiments 1-14, the peptide complex of any one of embodiments 15- 128, the peptide-active agent complex of any one of embodiments 129-137, or the pharmaceutical composition of embodiment 138 or embodiment 139, thereby treating the disease or condition.

EXAMPLES

[0438] The following examples are included to further describe some aspects of the present disclosure and should not be used to limit the scope of the invention.

EXAMPLE 1 Manufacture of Peptides

[0439] This example describes the manufacture of the peptides and peptide complexes described herein (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, SEQ ID NO: 1 - SEQ ID NO: 64, SEQ ID NO: 318 - SEQ ID NO: 390, SEQ ID NO: 457 - SEQ ID NO: 494, SEQ ID NO: 495 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526). Peptides derived from proteins were generated in mammalian cell culture using a published methodology. (A.D. Bandaranayke, C. Correnti, B.Y. Ryu, M. Brault, R.K. Strong, D. Rawlings. 2011. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Research. (39)21, el43).

[0440] The peptide sequence was reverse-translated into DNA, synthesized, and cloned in-frame with siderocalin using standard molecular biology techniques (M.R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). The resulting complex was packaged into a lentivirus, transduced into HEK-293 cells, expanded, isolated by immobilized metal affinity chromatography (IMAC), cleaved with tobacco etch virus (TEV) protease, and purified to homogeneity by reverse-phase chromatography. Following purification, each peptide was lyophilized and stored frozen.

EXAMPLE 2

EGF Variant Screening

[0441] This example describes the stepwise strategy of generating EGF variants capable of inhibiting EGFR and/or variants for incorporation into peptide complex (such as a selective depletion complex (SDC)).

[0442] FIG. 1 illustrates an EGFR-EGF complex showing EGFR ectodomains in their homodimeric, active conformation due to binding with EGF rendering each ectodomain capable of dimerization. Under basal conditions, EGFR Domain II is associated with membrane- proximal Domain IV (not shown). EGF binding to both EGFR Domain I and Domain III brings Domain II away from Domain IV and into a conformation capable of association with another Domain II. This crystal structure represents an active EGFR ectodomain homodimer (tetrameric due to the presence of two EGF molecules). Association of two EGFR molecules through this mechanism brings intracellular kinase domains (not shown) in close proximity, permitting crossphosphorylation and subsequent recruitment of growth signaling mediators like PI3K and SOS; these can drive oncogenic growth and other disease-causing pathways. Eliminating EGF binding to either Domain I and/or Domain III of EGFR in order to prevent receptor activation is therefore desirable in order to design an EGFR SDC or an EGFR inhibitor. Thus, use of an EGF variant in EGFR selective depletion is desirable via binding to EGFR without activation of growth signaling, necessitating interaction with one or the other, but not both, of Domains I and III.

[0443] EGF variants that bound EGFR Domain III but eliminated EGFR Domain I-binding were preferably selected since EGFR Domain III is present on the glioma driving variant EGFRvIII. Additionally, EGFR Domain III is known to lose EGF association at low pH, which can be advantageous for an EGFR SDC. To test if EGFRvIII variant could bind to wild type human EGF, 293F cells were grown in suspension culture in FreeStyle media and distributed into a 24- well suspension culture plate. One well was then transfected with a DNA construct driving surface expression of wild type human EGF (SEQ ID NO: 317). After 24 hours, cells were collected and incubated on ice with either biotinylated full length human EGFR ectodomain (full length EGFR) or biotinylated EGFRvIII ectodomain (EGFRvIII), along with streptavidin labeled with a dye that fluoresces in the APC channel, at 50 nM each. After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. Cells surface-displaying wild type human EGF could be stained with full length EGFR but not EGFRvIII, a variant that lacks Domain I (FIG. 2). Thus, wild type EGF did not appreciably bind to isolated EGFR Domain III (FIG. 2), necessitating identifying desired variants that potentially included improvement of EGF :EGFR Domain III binding in the absence of Domain I. The disruption of Domain I binding by mutated residues at that interface was tested. Additional Domain III affinity was engineered into the molecule by conventional affinity maturation. At the end, validation testing of the EGF variants for strong Domain III binding, lack of Domain I binding, and maintenance of natural pH-dependent release were performed.

[0444] FIG. 3 shows the stepwise flow chart used for conversion of EGF into a variant ready for incorporation into selective depletion complex (SDC) or use as a direct EGFR inhibitor, which were followed. An EGF variant capable of appreciable EGFR Domain III binding was first identified among variants derived from either Rosetta mutational binding improvement or sitesaturation mutagenesis, both of which were subjected to random mutagenesis. The resulting Stage 1 variant had improved EGFR binding overall but was still capable of growth signaling via dual Domain I/III interaction. To disrupt growth signaling, this Stage 1 variant was mutated to disrupt Domain I interacting residues. The resulting Stage 2 variant appreciably binds EGFR, but disruption of Domain I binding abrogated growth signaling capability. SDC incorporation requires sufficient EGFR binding to facilitate vesicular uptake, so further affinity maturation of this Stage 2 variant was performed through site-saturation mutagenesis and combination/permutation of enriched mutations. The resulting Stage 3 variant was tested for the maintenance of the pH-dependent release of wild type EGF; if this was lost, pH-dependent release was engineered into this Stage 3 variant by appropriate means (e.g., histidine substitution). The Final variant (which may or may not be identical to the Stage 3 variant, depending on whether or not pH maturation is required; for example, in the case of SEQ ID NO: 494, this additional pH maturation was not necessary) was incorporated into SDCs.

[0445] Prior to stage 1, models of published protein structures for the EGF:EGFR complexes were analyzed (FIG. 4), labeling the relevant domains as it related to EGF -variant design. This included modeling to determine EGFR-contacting residues of EGF that would have the greatest effect on binder maturation. On the Domain Ill-adjacent surface, these included residues where mutation could directly improve binding by better fitting into pockets on EGFR and/or creation of novel interactions like charge-charge salt bridges. For example, residues contacting Domain III (including, but not limited to, L15, R41, Q43, Y44, L47, K48, and E51) and neighboring residues were desirable to mutate in order to improve EGF:EGFR Domain III binding strength. Residues distal to the Domain III interface were also desirable to mutate, as such mutations can improve binding by altering overall EGF shape or rigidity. On the Domain I-adjacent surface, these included residues where the interface was to be disrupted to reduce or eliminate binding, either by inserting larger side chains to disrupt pocket fitting or removing elements that participate in pockets or salt bridge interactions. For example, residues contacting Domain I (including, but not limited to, M21, 123, A25, L26, K28, A30, N32, V35, 138, E40, and W49) were desirable to mutate in order to disrupt EGF:EGFR Domain I binding strength.

[0446] The first step, creation of EGF variants that bind to Domain III in isolation, was performed using EGFRvIII for binding assessment; this was to ensure that improvements to Domain III binding would not be dependent on Domain I for effect. A library containing hundreds of variants of human EGF was created using various design methods. First, the isolated structures of EGF and EGFR Domain III were analyzed using a Rosetta protein design script to identify residues on EGF whose mutation into other residues would result in computationally predicted strong binding. From these simulations, 488 unique EGF variants were selected. Mutations that appeared on at least 10% of these 488 high-scoring Rosetta- designed variants, which could therefore be considered highly favorable by Rosetta’s binding affinity calculations, include Q43I, Q43V, Q43W, Q43Y, K48N, K48T, K48A, K48L, and E51S. Second, wild type human EGF was subjected to site-saturation mutagenesis, wherein individual mutant alleles were created containing single mutations for each of the 47 non-Cys residues of EGF into each of the 18 other non-identical, non-Cys natural amino acids. This yielded 846 additional variants. EGF itself was also included in the library, for a final library size of 1335 EGF variants, and this library was also subjected to random mutagenesis via error- prone PCR prior to cloning. The mutagenized library was cloned into a vector for mammalian display of CDPs (fusion to a transmembrane tether via the N-terminus of the relevant EGF variant), converted into lentivirus, transduced into 293F cells at a multiplicity of infection (MOI) of ~1. EGFR binding of the pooled library was tested by staining the pool with either biotinylated control protein, biotinylated full length EGFR, or biotinylated EGFRvIII at 100 nM along with 100 nM streptavidin that fluoresces in the APC channel (approximately 647 nm excitation) for 10 minutes on ice. Cells were pelleted and resuspended in buffer containing DAPI, in order to exclude dead cells (DAPI+) from analysis. As shown in FIG. 5, flow cytometry analysis of the viable cell populations to assess GFP fluorescence (EGF variant expression) and APC channel fluorescence (target protein + streptavidin binding) showed that the library has a high proportion of full-length EGFR binding variants (~23% of GFP+ cells), with a much smaller proportion capable of showing appreciable binding to Domain III alone (EGFRvIII, <2% of GFP+ cells; i.e., variants whose EGFR binding is not dependent on also interacting with Domain I).

[0447] The EGF variant library in mammalian display vector was transduced into 293 T cells at an MOI of ~1 and incubated as a pool with biotinylated EGFRvIII and magnetic beads conjugated with anti-biotin antibodies. A magnet was then used to collect cells that accumulate EGFRvIII on their surface, removing non-magnetic cells. The resulting cells were cultured for 2 days and then stained with biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation) at 100 nM each for 15 minutes on ice. Cells were pelleted and resuspended in buffer containing DAPI, in order to exclude dead cells (DAPI+) from analysis. After performing flow cytometry analysis on the viable cell populations to assess GFP fluorescence (EGF variant expression) and APC channel fluorescence (target protein + streptavidin binding), GFP+ cells with APC channel fluorescence above background levels were flow sorted and collected. The EGF variant sequences within this population were PCR amplified, subcloned into a mammalian display vector for lentiviral transduction at an MOI of ~1 and subjected to the same staining and flow sorting for a further two rounds of flow sorting enrichment. As seen in FIG. 6, sequential enrichment of cells expressing surface displayed- EGFRvIII-binding EGF variants was achieved through the process described herein. By the end of this stage of screening campaign, EGF variants derived from either Rosetta design or saturation mutagenesis that were capable of binding EGFRvIII were surface displayed and had been enriched by magnetic sorting once and by flow sorting three times. These enriched EGF variant sequences were then cloned as singletons, each to be quantitatively assessed for EGFRvIII binding. [0448] EGF variants resulting from the EGFRvIII screen were PCR amplified and cloned into a mammalian display plasmid vector. Single transformants were picked as colonies, grown, and sequenced. 22 plasmids from transformed colonies each containing a unique EGF variant that was free of mutations that would disrupt function (e.g., cysteine mutations or premature stop codons) were transfected individually into wells of 293F cells in a suspension-growth 24 well plate. After 24 hours, cells were pelleted and stained with 50 nM each biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation). After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. Examples of single EGF variant staining capabilities upon transfection are shown in FIG. 7. One EGF variant did not bind EGFRvIII (e.g., SEQ ID NO: 320), but most did bind EGFRvIII (21 of 22, 8 of which are shown in FIG. 7). Two of the binders (SEQ ID NO: 319 and SEQ ID NO: 321) were based on Rosetta designs. A subpopulation of cells with prominent GFP expression was gated and fluorescence in these cells was measured, and this analysis allowed the identification of SEQ ID NO: 319 (a Rosetta design which incorporated 3 mutations; two were mutations frequently encountered among the Rosetta designs in this screen, K48T and E51S, with an additional V34S mutation acquired during random mutagenesis) as the lead variant (also herein “EGF84” for the 84^th clone from the screen tested). Thus, the experiments showed successful generation of EGF variants capable of binding to EGFRvIII alone, demonstrating improved binding to EGFRvIII compared to parental EGF.

[0449] The screen for improved EGFRvIII binding also identified mutations to residues at the Domain I interface that were tolerated in screens for improved Domain III binding (FIG. 8). The 21 EGFRvIII -binding EGF variants all contained at least one mutation, and many of these mutations were in sites defined as Domain I-contacting mutations. Studying these overlapping sites allowed the identification of mutations to Domain I-contacting residues that do not prevent Domain III binding. Four such mutations are M21R (present in SEQ ID NO: 378), A30W (present in SEQ ID NO: 371), I38D (present in SEQ ID NO: 376), and W49R (present in SEQ ID NO: 383, SEQ ID NO: 384, and SEQ ID NO: 385). Three of these mutations (M21R, I38D, and W49R) replaced hydrophobic residues with polar charged residues, potentially disrupting hydrophobic interactions strengthening the EGF:EGFR Domain I interaction. The other mutation, A30W, replaced a small side chain with a large, bulky side chain, potentially disrupting EGF:EGFR Domain I interaction by steric hindrance. These four mutations were considered as candidates for incorporation into an EGF variant to disrupt Domain I-binding and eliminate EGFR activation from the candidate EGFR SDC. [0450] The identified mutations were studied in the context of the crystal structure to verify that they are predicted to disrupt Domain I-b inding (FIG. 9). Close study of the interactions between EGF and EGFR, based on the publicly available crystal structure deposited into the RCSB as PDB ID: 1IVO, revealed that all four potential Domain I-disrupting substitutions either introduce a charged amino acid where otherwise the nonpolar residue closely abuts EGFR Domain I (M21R, 138D, and W49R) or introduces a bulky side chain whose most energetically- favorable conformation would spatially overlap with that of EGFR Domain I (A30W). In summary, based on structural analysis, all four candidate mutations can disrupt EGF:EGFR Domain I binding.

[0451] Variants of an exemplary EGFRvIII-binding EGF variant (SEQ ID NO: 319) were designed incorporating M21R, A30W, I38D, or W49R mutations (SEQ ID NO: 388 - SEQ ID NO: 390 and SEQ ID NO: 457), alongside one incorporating all 4 mutations (SEQ ID NO: 458). They were individually cloned into a mammalian surface display vector and transfected individually into wells of 293F cells in a suspension-growth 24 well plate. After 24 hours, cells were pelleted and stained with 50 nM each biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation). After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. As shown in FIG. 10, EGFRvIII binding was observed for all five variants, with the highest staining seen by SEQ ID NO: 458 which incorporated all four mutations, in spite of the disruption in EGF:EGFR Domain I binding for each variant.

[0452] One variant at this stage, EGF84v9 (designated as SEQ ID NO: 458), has 7 total mutations from wild type human EGF and 4 mutations from EGF84. Human EGF (SEQ ID NO: 317) and the EGF variant, EGF84v9 (SEQ ID NO: 458) were individually cloned into a mammalian surface display vector and transfected individually into wells of 293F cells in a suspension-growth 24 well plate. After 24 hours, cells were pelleted and stained with either 50 nM biotinylated full length EGFR or 50 nM EGFRvIII along with 50 nM streptavidin that fluoresces in the APC channel (approximately 647 nm excitation). After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. As shown in FIG. 11, EGF demonstrated binding to full length EGFR but not EGFRvIII, while EGF84v9 (SEQ ID NO: 458) displayed very strong binding to EGFRvIII but weaker binding to full length EGFR than to EGFRvIII, confirming disruption of Domain I binding; the weaker staining may indicate some steric disruption of EGF variant:EGFR Domain III binding when Domain I is both present and not specifically binding to the EGF variant in an energetically-favorable way. Taken together, this example demonstrated the successful development of a variant of EGF that binds Domain III without Domain I, and is not favorably interacting with Domain I. Such a variant would not promote EGFR signaling as it would not be capable of allowing Domain II to dissociate from Domain IV. As a result, this EGF variant would not promote the oncogenic potential and growth of cancer cells. Although the EGF variants herein bind EGFR, they would disrupt the dimerization of EGFR, and hence disrupt, or reduce, or ablate the promotion of growth and hence disrupt or reduce, or ablate the oncogenic potential of the EGFR on cells.

EXAMPLE 3

Affinity Maturation of an EGF Variant (EGF84v9)

[0453] This example describes the affinity maturation of an EGF variant, using EGF84v9 (SEQ ID NO: 458) as the parental sequence. After site-saturation mutagenesis library (containing all non-cysteine substitution single mutants of a parental sequence and the parental sequence itself) cloning, lentiviral transduction, cell staining, flow sorting to enrich for variants that bind tightly to EGFRvIII at pH 7.4 while also releasing it at pH 5.5, and sequencing to identify mutations that convey these advantageous properties, 36 variants of EGF84v9 (SEQ ID NO: 458) were designed that contained at least 4 of 6 highly enriched mutations (DI 1R, I23S, V35E, S51P, L52E, and R53E) and/or His substitutions (S51H, L52H, or R53H) or truncations within the terminal 3 amino acids (deleting R53 alone, both of L52 and R53, or all three of S51, L52, and R53) (shown in FIG. 12). All of these mutant variants, along with EGF84v9 (SEQ ID NO: 458) itself, were individually cloned into a mammalian surface display vector and transfected individually into wells of 293F cells in a suspension-growth 24 well plate. After 24 hours, cells were pelleted and stained with 20 nM each biotinylated EGFRvIII and streptavidin that fluoresces in the APC channel (approximately 647 nm excitation). After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. As shown in FIG. 13, while all of the variants derived from site-saturation mutagenesis screening demonstrated improved EGFRvIII staining compared to the parental EGF84v9 (SEQ ID NO: 458), four were selected for further analysis due to high EGFRvIII staining, SEQ ID NO: 477, SEQ ID NO: 481, SEQ ID NO: 493, and SEQ ID NO: 494. [0454] The four affinity-matured variants of EGF84v9 (SEQ ID NO: 458), including SEQ ID NO: 477, SEQ ID NO: 481, SEQ ID NO: 493, and SEQ ID NO: 494, along with EGF84v9 (SEQ ID NO: 458) itself, were further tested to confirm maintenance of low-pH release. They were individually cloned into mammalian surface display vector and transfected individually into wells of 293F cells in a suspension-growth 24 well plate. After 24 hours, cells were pelleted and stained with 20 nM each 6xHis-tagged EGFRvIII and anti-6xHis antibody that fluoresces in the APC channel (approximately 647 nm excitation); this variation on co-stain (anti-6xHis instead of streptavidin) is both to better permit dissociation (streptavidin confers tetravalent avidity to cell staining, while anti-6xHis (“6xHis” disclosed as SEQ ID NO: 142) only confers bivalent avidity), and because bivalent staining better simulates the EGFR selective depletion complex (SDC) designs that use bivalent Fc fusion proteins. After 30 minutes, cells were pelleted and resuspended in either a citrate/phosphate buffer of pH 5.5 or a PBS buffer of pH 7.4. After 10 minutes, cells were pelleted and resuspended in a buffer containing DAPI for flow cytometry analysis of viable cells. A subpopulation of cells with defined GFP expression was gated and fluorescence in these cells was measured. As shown in FIG. 14, all variants demonstrated substantial reduction of EGFRvIII staining upon pH 5.5 rinse compared to pH 7.4 rinse, but SEQ ID NO: 494 had the highest staining retention after pH 7.4 rinse. After following the design steps illustrated in the flow chart shown in FIG. 3, including Rosetta design, Domain I interface disruption, affinity maturation, and confirmation of pH dependent binding, SEQ ID NO: 494 was identified as the EGF variant to be advanced for EGFR SDC incorporation. It was the 36th variant tested; including the parental sequence, it was EGF84v9 (SEQ ID NO: 458) variant 37, or EGF84v9.37 (SEQ ID NO: 494). EGF84v9.37 (SEQ ID NO: 494) is 50 AA in length; wild type human EGF is 53 AA. Including this truncation, EGF84v9.37 (SEQ ID NO: 494) has 6 mutations differentiating it from EGF84v9 (SEQ ID NO: 458) and 12 mutations differentiating it from wild type human EGF (SEQ ID NO: 317) (one of the mutations between EGF and EGF84 is in this truncated region, so it is not counted twice upon truncation). EGF84v9.37 (SEQ ID NO: 494) has 77% (41/53) amino acid identity with wild type human EGF (SEQ ID NO: 317).

EXAMPLE 4

EGFR Selective Depletion Complex (SDC) Design with TfR Binders

[0455] This example describes certain selective depletion complexes (SDCs) based on the identified EGF variants identified in EXAMPLE 2 and EXAMPLE 3, such as EGF variants of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. FIG. 15 illustrates various protein domains or linkers that, when joined into a molecule containing at least one EGFR-b inding domain and at least one TfR-binding domain, constitute an EGFR SDC. These molecules are designed with an EGF variant fused to the protein via the N-terminus of the EGF variant, keeping the C-terminus of the EGF variant free. The C terminus of EGF84v9.37 (SEQ ID NO: 494) is adjacent to the surface of Domain III, so fusion to this site could disrupt interactions with Domain III. Meanwhile, the N-terminus of EGF has no apparent interaction with EGFR (demonstrated by the co-crystal structure of PDB ID: 1IVO, where the N-terminal four residues of EGF do not resolve in the crystal structure). All seven molecules shown in FIG. 15 contain an Fc domain (homodimeric or heterodimeric knob-and-hole) and one or two domains constituting EGF84v9.37 (SEQ ID NO: 494). Six also contain one or two domains constituting TfR-binding CDPs of SEQ ID NO: 96, making EGFR SDC candidate molecules (SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 503 paired with SEQ ID NO: 504, SEQ ID NO: 503 paired with SEQ ID NO: 505, and SEQ ID NO: 504 paired with SEQ ID NO: 506). The SDC candidate molecules are distinguished by the linker between the TfR-binding domain and the EGF variant domain, and also variation in valence of EGFR or TfR binding capabilities. Linkers between the Fc domain and the TfR-binding domain could also vary; the example here include molecules using a linker comprising SEQ ID NO: 223 to separate the Fc domain and the TfR-binding domain, but an SDC could also use a linker comprising any of SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541, or any other linker sequence, to separate the Fc domain and the TfR-binding domain. A control SDC without TfR-binding capabilities is included (SEQ ID NO: 498). Though the control SDC of SEQ ID NO: 498 does not have TfR binding capabilities it does have EGFR binding capabilities and is predicted to bind EGFR without facilitating growth-signal-driving dimerization which could render SEQ ID NO: 498 into an effective EGFR inhibitor. SEQ ID NO: 498, therefore, may be an EGFR inhibitor and may not be an SDC.

EXAMPLE 5

EGFR Selective Depletion Complex (SDC) Design with PD-L1 Binders

[0456] This example describes certain selective depletion complexes (SDCs) based on the identified EGF variants identified in EXAMPLE 2 and EXAMPLE 3, such as EGF variants of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. FIG. 16 illustrates various protein domains or linkers that, when joined into a molecule containing at least one EGFR-binding domain and at least one PD-L1 -binding domain, constitute an EGFR SDC. PD-L1 is expressed on many cancer cells, and its trafficking between the surface and intracellular compartments shares many characteristics with TfR trafficking, which could permit such PD-L1 -binding SDCs to drive EGFR depletion in cancer cells expressing PD-L1. These molecules are designed with an EGF variant fused to the protein via the N-terminus of the EGF variant, keeping the C-terminus of the EGF variant free. The C terminus of EGF84v9.37 (SEQ ID NO: 494) is adjacent to the surface of Domain III, so fusion to this site could disrupt interactions with Domain III. Meanwhile, the N-terminus of EGF has no apparent interaction with EGFR (demonstrated by the co-crystal structure of PDB ID: 1IVO, where the N-terminal four residues of EGF do not resolve in the crystal structure). The six molecules shown in FIG. 16 contain an Fc domain (homodimeric or heterodimeric knob-and-hole) and one or two domains constituting EGF84v9.37 (SEQ ID NO: 494) and one or two domains constituting PD- Ll-binding CDPs of SEQ ID NO: 187, making EGFR SDC candidate molecules (SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 515 paired with SEQ ID NO: 516, SEQ ID NO: 515 paired with SEQ ID NO: 505, and SEQ ID NO: 506 paired with SEQ ID NO: 516). The SDC candidate molecules are distinguished by the linker between the PD-L1 -binding domain and the EGF variant domain, and also variation in valence of EGFR or PD-L1 binding capabilities. Linkers between the Fc domain and the PD-Ll-binding domain could also vary; the example here include molecules using a linker comprising SEQ ID NO: 223 to separate the Fc domain and the PD-Ll-binding domain, but an SDC could also use a linker comprising any of SEQ ID NO: 538, SEQ ID NO: 540, or SEQ ID NO: 541, or any other linker sequence, to separate the Fc domain and the PD-Ll-binding domain.

EXAMPLE 6 EGF Variants and EGFR Disruptors

[0457] This example describes certain EGF variants and EGF variant active agent complexes based on the identified EGF variants identified in EXAMPLE 2 and EXAMPLE 3. Samples were analyzed by Western blot to assess disruption of EGFR signaling by an EGF -variant selective depletion complex (SDC), as well as the absence of EGFR activation by molecules that incorporate the EGF variant (designed to bind EGFR but not activate it). A549 cancer cells were grown until approximately 30% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were left untreated or dosed with either the SDC comprising SEQ ID NO: 495 or the control comprising SEQ ID NO: 498 at 10 nM and incubated for a further 24 hours. After 24 hours, and without exchanging media, cells were either left as-is or exposed to 50 ng/mL EGF (SEQ ID NO: 317). Cells were then lysed in buffer containing protease/phosphatase inhibitors. Lysate protein content was quantitated by bicinchoninic acid (BCA) assay and was ran on an SDS-PAGE gel prior to electrophoretic transfer to a polyvinylidine difluoride (PVDF) membrane and subsequent blocking and blotting with antibodies against actin, total EGFR, or phospho-Y1068 (pY1068) EGFR, the latter being a marker for ligand-activated EGFR. A fluorescent secondary antibody provides signal that can be read on a scanner. The blots demonstrate five phenomena, as shown in FIG. 19, and described below. First, untreated cells have baseline minimal pY1068 EGFR (ligand-activated EGFR) levels in the absence of EGF, as seen in Lane 1 of FIG. 19. Second, EGF induces EGFR Y1068 phosphorylation, as expected, as seen in Lane 2, of FIG. 19. Third, the SDC of SEQ ID NO: 495 (which contains an EGFR-binding EGF variant (SEQ ID NO: 494)) substantially reduces EGF- induced EGFR Y1068 phosphorylation, as would be predicted by this SDC that has been shown to deplete surface EGFR, as seen in comparing Lanes 2 and 4 of FIG. 19. Fourth, that this reduction of ligand-activated EGFR is enhanced by the TfR-binding in the SDC, by showing that the control molecule lacking TfR-binding that uses the same EGFR-binding EGF variant (SEQ ID NO:494) as the SDC (SEQ ID NO: 498) does not reduce Y1068 phosphorylation to the same degree, as seen in comparing Lanes 4 and 6, of FIG. 19. Fifth, both SEQ ID NO: 495 and SEQ ID NO: 498 contain EGF variants, but neither produces a significant EGFR pY1068 signal on its own, as seen in Lanes 3 and 5 of FIG. 19. This last observation validates the design process wherein the EGF variants are engineered to bind EGFR without facilitating its dimerization/activation. Due to the disruption of EGFR dimerization, the EGF variants can be used to disrupt EGFR homodimerization (or other multimerization) and/or EGFR-based cell signaling, resulting in a therapeutic intervention directly on cancer cells expressing EGFR or indirectly through reducing cell signaling and other moieties activated by EGF-responsive cancer cells. Such EGF variants can act alone or in combination with an active agent described herein.

EXAMPLE 7

Selective Depletion of EGFR via TfR-mediated Endocytosis

[0458] This example describes selective depletion of EGFR via TfR-mediated endocytosis. The selective depletion complexes (SDC) were tested for their ability to eliminate surface EGFR in cells and promote uptake of soluble EGFR into the cell. Selective depletion complexes of SEQ ID NO: 495, which have a TfR-binding peptide of SEQ ID NO: 96 and an EGFR target-binding peptide of any of SEQ ID NO: 494, were tested.

[0459] The extent of elimination of surface EGFR in cells by the SDC was evaluated by analyzing samples by flow cytometry to measure the surface EGFR under different SDC concentrations. A549 cancer cells were grown until approximately 30% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were then left untreated (“Untreated”) or dosed with the SDC comprising SEQ ID NO: 495 at 2 nM, 10 nM, 50 nM, or 200 nM and incubated for a further 24 hours (untreated or in the presence of the SDC). After 24 hours, cells were rinsed, collected, stained with an antibody vs EGFR and a fluorescent co-stain, and subjected to flow cytometry to quantitate fluorescent signal corresponding to the amount of surface EGFR per cell in the population. The data for each population were averaged and plotted relative to the untreated population as provided in FIG. 17A. As seen in FIG. 17A, the doses of the SDC comprising between 2 nM and 200 nM were capable of substantially reducing the amount of surface EGFR after a 24 hour incubation.

[0460] The extent and duration of elimination of surface EGFR in the cells by an SDC was evaluated by analyzing samples by flow cytometry to measure the surface EGFR under different SDC treatment regimens. A549 cancer cells were grown until approximately 20% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. Cells were then left untreated or dosed with 10 nM of the SDC comprising SEQ ID NO: 495 and incubated for a further 24 hours. After 24 hours, the media in the wells was replaced with fresh media. Three treatments were then performed and analyzed over the next 24 hours (which treatments are summarized in the table in FIG. 17B): (1) the cells that had been subjected to treatment with the SDC comprising SEQ ID NO: 495 for 24 hours were then left untreated for an additional 24 hours (referred to as the “Withdrawal” sample, as it was subjected to 24 hour treatment with the SDC followed by a 24 hour in untreated media (that is, treatment withdrawal of the SDC)), (2) previously-untreated cells were dosed with 10 nM of the SDC comprising SEQ ID NO: 495 for 24 hours (referred to as the “24 hr” sample, as its surface EGFR was analyzed immediately after a 24 hour SDC treatment), and (3) a third sample (“Untreated”) that was left untreated during the previous 24 hours was still left untreated for an additional 24 hours. After this second 24 hour incubation, cells in all samples were rinsed, collected, stained with an antibody vs EGFR and a fluorescent co-stain, and subjected to flow cytometry to quantitate fluorescent signal corresponding to the amount of surface EGFR per cell in the population. The data for each population were averaged and plotted relative to the untreated population as provided in FIG. 17B. As shown in FIG. 17B, 10 nM of the SDC comprising SEQ ID NO: 495 was capable of substantially reducing the amount of surface EGFR after a 24 hour incubation, whether measured immediately, or after a 24 hour drug withdrawal period. The latter (after the 24 hour drug withdrawal) is a demonstration of catalytic activity of the SDC, as surface EGFR would have been expected to have completely recovered in the cells after 24 hour based on synthesis of new EGFR. The fact that EGFR stayed suppressed after drug withdrawal strongly suggests that the SDC comprising SEQ ID NO: 495 remained associated with the cells and continued clearing EGFR even after SDC was removed from the media. The level of EGFR at the cell surface, if recovered, would be expected to be similar to levels of the untreated control. In other words, the SDC comprising SEQ ID NO: 495 remains catalytically active in facilitating surface depletion of EGFR, as it retains depletion activity after soluble SDC is removed from the media because the remaining cell-associated SDC molecules continue to catalytically facilitate surface EGFR depletion over the ensuing 24 hours. A non-catalytic molecule facilitating surface uptake or depletion would lose its activity after soluble SDC removal from the media, as any cell- associated SDC molecules would only facilitate uptake of their then currently-bound EGFR but nothing more, allowing the cell to return to recovered and steady state EGFR levels within 24 hours.

[0461] The uptake of soluble EGFR into the cells was evaluated by analyzing samples by flow cytometry to measure the soluble EGFR uptake under different SDC treatment regimens. Hl 975 cancer cells were grown until approximately 40% confluence in RPMI media with 10% FBS and antibiotic/antimycotic supplementation. After this point, cells underwent one of three treatments to assess uptake of soluble EGFRvIII (sEGFR) LEEKKGNYWTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDS LSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW PENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKNLCY ANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVS RGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPH CVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS; SEQ ID NO: 527). Each of the three treatments are summarized in the table in FIG. 18. Treatment (1), referred to as “No SDC” in FIG. 18, incubated the cells for two hours in media with 20 nM sEGFR and 20 nM unlabeled monovalent streptavidin. After this 2 hour treatment, cells are rinsed twice with PBS and then incubated for 24 hours in media with 10 nM sEGFR and 10 nM fluorescent streptavidin. Any accumulation of fluorescent signal, as shown in FIG. 18, in this period shown was due to passive uptake of media components and not due to SDC because the SDC was never present. Treatment (2), referred to as “SEQ ID NO: 495 Pre-treat” in FIG. 18, incubated the cells for 2 hours in media with 20 nM sEGFR, 20 nM unlabeled monovalent strepatavidin, and 5 nM of the SDC comprising SEQ ID NO: 495. This treatment first exposed cells to sEGFR-saturated SDC molecules. After this 2 hour treatment, cells were rinsed twice with PBS and then incubated for 24 hours with 10 nM sEGFR and 10 nM fluorescent streptavidin to assess entry of the fluorescent sEGFR into the cells. If the SDCs from the initial 2-hour treatment were not catalytic, the bound, unlabeled sEGFR (on the sEGFR- saturated SDC molecules) would be the only sEGFR they would be capable of entering into the cell via TfR-associated endocytosis, possessing no further activity and driving no entry of later applied fluorescent sEGFR into the cells. The subsequent 24 hour incubation with sEGFR and fluorescent streptavidin would not yield fluorescent signal accumulation beyond that seen by the SDC-free incubation (“No SDC”). Instead, as shown in FIG. 18, an increase in accumulated fluorescent signal in the “SEQ ID NO: 495 Pre -treat” sample was seen, which required an SDC with catalytic activity to facilitate increased uptake of later applied fluorescent sEGFR into the cells. Treatment (3), referred to as “SEQ ID NO: 495 24 hr” in FIG. 18, was the same treatment as “SEQ ID NO: 495 Pre-treat”, except the second incubation (24 hours with 10 nM sEGFR and 10 nM fluorescent streptavidin) also included 5 nM SDC comprising SEQ ID NO: 495. The inclusion of SDC in the second incubation was to demonstrate the maximum fluorescent signal uptake, as shown in FIG. 18, of fluorescent sEGFR into the cells that 5 nM SDC induced under these conditions.

[0462] The SDC of Seq ID NO: 495 was shown to deplete EGFR from the cell surface, as shown in FIG. 17A and FIG. 17B, and also increase the cellular uptake of soluble EGFR (sEGFR, SEQ ID NO: 527), as shown in FIG. 18.

EXAMPLE 8 EGF Variants and EGFR Disruptors

[0463] This example describes certain EGF variants and EGF variant active agent complexes based on the identified EGF variants identified in EXAMPLE 2 and EXAMPLE 3, such as EGF variants of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. The EGF variant of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494 binds EGFR and disrupts the dimerization of EGFR. The EGF variant prevents binding of EGF and inhibits activation of EGFR. Thus, binding of the EGF variant to EGFR disrupts, reduces, or ablates the promotion of growth and/or the oncogenic potential of the EGFR on cells. The EGF variants would not promote the oncogenic potential and growth of cancer cells. Due to the disruption of EGFR dimerization, the EGF variants can be used to disrupt EGFR homodimerization (or other multimerization) and/or EGFR-based cell signaling, resulting in a therapeutic intervention directly on cancer cells expressing EGFR or indirectly through reducing cell signaling and other moieties activated by EGF-responsive cancer cells. Such EGF variants can act alone or in combination with an active agent described herein.

EXAMPLE 9

Treatment of Cancer Using EGF Variants and EGFR Disruptors

[0464] This example describes certain EGF variants and EGF Variant active agent complexes based on the identified EGF variants identified in EXAMPLE 2 and EXAMPLE 3, such as EGF variants of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494. Upon administration to a subject having cancer, the EGF variant of SEQ ID NO: 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494 disrupts the dimerization of EGFR. The EGF variant prevents binding of EGF and inhibits activation of EGFR. Thus, binding of the EGF variant to EGFR disrupts, reduces, or ablates the promotion of growth and the oncogenic potential of the EGFR on cells. The EGF variants would not promote the oncogenic potential and growth of cancer cells. Due to the disruption of EGFR dimerization, the EGF variants can be used to disrupt EGFR homodimerization (or other multimerization) and/or EGFR-based cell signaling, resulting in a therapeutic intervention directly on cancer cells expressing EGFR or indirectly through reducing cell signaling and other moieties activated by EGF-responsive cancer cells. Administration of the EGF variant inhibits growth of cancer cells, thereby treating or preventing cancer in the subject. The EGF variants can be administered alone or in combination with an active agent described herein to treat the cancer.

EXAMPLE 10

Selective Depletion of EGFR via TfR-mediated Endocytosis

[0465] This example describes selective depletion of EGFR via TfR-mediated endocytosis. A composition containing a TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) fhsed, complexed, or conjugated to an EGFR target-binding peptide of any of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494 is contacted to cells expressing TfR. Optionally, the composition contains a selective depletion complex of any of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526. Optionally, The TfR-binding peptide binds TfR with high affinity at both physiologic extracellular pH (such as at pH 7.4) and at endosomal pH (such as at pH 5.5), and the target-binding peptide binds to a surface target molecule with higher affinity at physiologic extracellular pH and with lower affinity at endosomal pH. Upon contact, the TfR- binding peptide binds to TfR on the cell surface, and the target-binding peptide binds to EGFR on the cell surface. The composition containing the TfR-binding peptide and the target-binding peptide is endocytosed via TfR-mediated endocytosis along with the TfR and the bound EGFR, thereby depleting the target EGFR. As the endosomal compartment acidifies, the EGFR is optionally released from the target-binding peptide. The EGFR is then optionally degraded in a lysosomal compartment and further depleting the target, and the complex is optionally recycled to the cell surface along with the TfR.

EXAMPLE 11

Selective Depletion of EGFR via PD-Ll-mediated Endocytosis

[0466] This example describes selective depletion of EGFR via PD-Ll-mediated endocytosis. A composition containing a PD-Ll-binding peptide (e.g., any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241) fused, complexed, or conjugated to an EGFR target-binding peptide of any of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494 is contacted to cells expressing PD-L1.

Optionally, the composition contains a selective depletion complex of any of SEQ ID NO: 511 — SEQ ID NO: 526. Optionally, The PD-Ll-binding peptide binds PD-L1 with high affinity at both physiologic extracellular pH (such as at pH 7.4) and at endosomal pH (such as at pH 5.5), and the target-binding peptide binds to a surface target molecule with higher affinity at physiologic extracellular pH and with lower affinity at endosomal pH. Upon contact, the PD-Ll- binding peptide binds to PD-L1 on the cell surface, and the target-binding peptide binds to EGFR on the cell surface. The composition containing the PD-Ll-binding peptide and the target-binding peptide is endocytosed via PD-Ll-mediated endocytosis along with the PD-L1 and the bound EGFR, thereby depleting the target EGFR. As the endosomal compartment acidifies, the EGFR is optionally released from the target-binding peptide. The EGFR is then optionally degraded in a lysosomal compartment and further depleting the target, and the complex is optionally recycled to the cell surface along with the PD-L1. EXAMPLE 12

Extension of Peptide Plasma Half-life Using Serum Albumin-binding Peptide Complexes [0467] This example demonstrates a method of extending the serum or plasma half-life of a peptide using serum albumin-binding peptide complexes as disclosed herein. A peptide or peptide complex having a sequence of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494, SEQ ID NO: 499 - SEQ ID NO: 501, or SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 507, SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526 is modified in order to increase its plasma half-life. The peptide and the serum half-life extending moiety are fused recombinantly, chemically synthesized as a single fusion, separately recombinantly expressed and conjugated, or separately chemically synthesized and conjugated. Fusing the peptide to a serum albumin-binding peptide extends the serum half-life of the peptide complex. The peptide or peptide complex is conjugated to a serum albumin-binding peptide, such as SA21 (SEQ ID NO: 178). Optionally, the peptide fused to SA21 has a sequence of any one of SEQ ID NO: 181 or SEQ ID NO: 184. Optionally, the peptide fused to SA21 is linked to SA21 via a peptide linker having a sequence of SEQ ID NO: 179. The linker having a sequence corresponding to SEQ ID NO: 179 links two separately functional CDPs to incorporate serum half-life extension function into the peptide or peptide complex. The linker having a sequence corresponding to SEQ ID NO: 179 enables SA21 to cyclize without steric impediment from either member of the peptide complex. Alternatively, conjugation of the peptide to an albumin binder, such as Albu-tag or a fatty acid, such as a CM-CIS fatty acid or palmitic acid, is used to extend plasma half-life. Plasma half-life is also optionally extended as a result of reduced immunogenicity by using minimal non-human protein sequences.

EXAMPLE 13

Linkers for Fusion, Complexation, or Conjugation and Half-life Extension of Targetbinding Peptides and TfR-binding Peptides

[0468] This example describes linkers for fusion, complexation, or conjugation and optionally for half-life extension of peptide complexes. A TfR-binding peptide (e.g., any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64) or a PD-Ll-binding peptide (e.g., any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241) is fused, linked, complexed, or conjugated to an EGFR target-binding peptide (e.g., any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) via a linker to form a peptide complex. The peptides can be fused via a DkTx peptide (SEQ ID NO: 139, KKYKPYVPVTTN) from native CDP dimer. The DkTx peptide linker is from a native knottin-knottin dimer from the Tau- theraphotoxin-Hsla, also known as DkTx (double-knot toxin), in Haplopelma schmidti.

Natively, the DkTx linker separates two independently folding CDP domains and is well-suited for maintaining the function of the two dimerizing CDPs. The peptides can be fused via a poly- GlySer linker such as (SEQ ID NO: 138, GGGSGGGSGGGS), containing varying lengths of glycines interspaced by serines for solubility. The peptides can be fused via a human IgG linker with a Cys-to-Ser mutation at position 5 (SEQ ID NO: 140, EPKSSDKTHT) to prevent crosslinking during secretion. A peptide linker for dimerizing two peptides optionally has the following properties: 1) the linker does not disturb the independent folding of the peptide domains, 2) the linker provides sufficient length to the mature molecule so as to facilitate contact between the target molecule and the receptor, 3) the linker does not negatively impact manufacturability (synthetic or recombinant) of the peptide complex, and 4) the linker does not impair any required post-synthesis chemical alteration of the peptide complex (e.g., conjugation of a fluorophore or albumin-binding chemical group).

[0469] CDPs (or other protein-based target-engaging modalities) can also be dimerized using immunoglobulin heavy chain Fc domains. These are commonly used in modem molecular medicine to dimerize functional domains, either based on antibodies or other otherwise soluble functional domains. The target-binding peptide can be non-covalently linked to the TfR-binding peptide via an IgG-based Fc domain. An Fc domain can be used to homo- or hetero-dimerize functional domains and to impart serum half-life extension via a domain that interacts with the recycling Fc receptor (FcRn). Dimerization can be homodimeric if the Fc sequences are native, but if one wants to drive heterodimer formation, the Fc can be mutated into “knob-in-hole” format, where one Fc CH3 contains novel residues (knob) designed to fit into a cavity (hole) on the other Fc CH3 domain. Through this process, knob+knob dimers are highly energetically unfavorable. Hole+hole dimers can be formed, but if a purification tag is added specifically to the “knob” side, hole+hole dimers can be excluded, ensuring that only knob+hole dimers are purified. Fc domains can separately be used as a recycling receptor-engaging domain, so use of Fc for dimerization can enhance peptide complex recycling or selective degradation complex. [0470] Peptide complexes can be further functionalized and multimerized by adding a third (or more) functional domain. In this example, an albumin-binding domain from a Finegoldia magna peptostreptococcal albumin-binding protein (SEQ ID NO: 192) is shown, as it is a simple three- helical structure that would be unlikely to disturb the independent folding of the other CDP domains. Such added functional domains could be included in any orientation relative to the peptide binding domains. An albumin binding domain (e.g., a peptide of SEQ ID NO: 178 or SEQ ID NO: 192) can be fused to the receptor-binding peptide, the target-binding peptide, or both. The albumin binding domain can include a peptide linker (e.g., any one of SEQ ID NO: 129 - SEQ ID NO: 141 or SEQ ID NO: 195 - SEQ ID NO: 218). Addition of the albumin binding domain can increase the serum half-life of a peptide complex.

EXAMPLE 14

Delivery of Selective Depletion Complexes or EGF Variants using Gene Therapy or Cell Therapy

[0471] This example describes delivery of selective depletion complexes using an oncolytic herpes simplex virus. A gene encoding expression and secretion of a selective depletion complex (e.g., a selective depletion complex of any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526) or an EGF variant (e.g., an EGF variant of any one of SEQ ID NO: 318 — SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) or an EGF variant peptide complex (e.g., SEQ ID NO: 498 or SEQ ID NO: 502) is introduced to a target cell using an oncolytic herpes simplex virus (oHSV) vector. For gene therapy, the target cell is a cell within a patient. For cell therapy, a target cell is a patient cell that has been collected and is re-introduced into the patient after modification with the viral vector. The oHSV infects cancer cells, and cancer cells that are not killed by the virus express and secrete the selective depletion complex. The remaining cells modify the tumor microenvironment to suppress immune activity against the cancer cells. Selective depletion complexes are secreted from the tumor cells in situ and act on the cancers are directed against immunosuppressive factors on T cells or in the tumor microenvironment.

[0472] Alternatively, selective depletion complexes that modify tumor or T-cell activity could are engineered into CAR-T cells or other cellular therapies. CAR-T cells are already being specialized through genetic modification to target tumor tissue, killing tumor cells that carry cell surface markers targeted by the expressed chimeric antigen receptors (CAR). If supplemental activity is desired, such as suppression of regulatory, immunosuppressive signaling present in these tumors, the CAR-T cell is engineered to also secrete selective depletion complexes that suppress regulatory, immunosuppressive signaling. EXAMPLE 15

Treatment of Cancer by Selectively Depleting EGER

[0473] This example describes treatment of cancer by selectively depleting EGFR. A selective depletion complex containing an EGFR-binding peptide, which is optionally pH dependent, such as that of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494, and a TfR-binding peptide, such as that of SEQ ID NO: 96, or alternatively, a PD-Ll-binding peptide such as that of any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241, or a selective depletion complex of any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526. is administered to a subject having an EGFR positive cancer. The subject may be a human, a non-human primate, a mouse, a rat, or another species. A selective depletion complex may be administered subcutaneously, intravenously, intramuscularly, interperitoneally, or by another route. It may be administered 1, 2, 3, 4, 5, 10, or more times and at a frequency of 1, 2, 3, 4, 5, 6, or 7 times per week or every other week or every third week, or monthly or every other month or every third month or less frequently. The EGFR positive cancer is non-small-cell lung cancer, head and neck cancer, glioblastoma, metastatic brain cancer, primary brain cancer, EGFRVIII-driven brain cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, TKI-resistant cancer, osimertinib-resistant cancer, gefitinib-resistant cancer, erlotinib-resistant cancer, cetuximabresistant cancer, necitumumab-resistant cancer, or panitumumab-resistant cancer. Optionally, the cancer overexpresses EGFR, is KRAS mutant, has a KRAS G12S mutation, has a KRAS G12C mutation, has PTEN loss, has an EGFR exonl9 deletion, has an EGFR L858R mutation, has an EGFR T790M mutation, has a cetuximab-resistant EGFR, has a panitumumab-resistant EGFR, has a mutant PIK3CA, has a TP53 R273H mutation, has PIK3Ca amplification, has PIK3CA G118D, has TP53 R273H, has a C79X mutation, a G724S mutation, an L718Q mutation, or an S768I mutation. The selective depletion complex binds to EGFR and TfR on the surface of an EGFR positive cancer cell, and the ternary complex of the selective depletion complex, EGFR, and TfR is endocytosed, thereby depleting surface EGFR. The EGFR may be released upon acidification in the endosome and degraded, thereby further depleting EGFR. The TfR and selective depletion complex are optionally recycled to the cell surface. Depletion of EGFR reduces pro-growth signaling in the cancer cell, slowing cancer growth or metastases, thereby treating the cancer. Depletion of EGFR may cause senescence in the cancer cells, may reduce the viability of the cancer cells, and may make the cancer cells more vulnerable to immune- mediated attack and clearance. Optionally, because the selective depletion complex targets cells that overexpress both EGFR and TfR, the skin toxicity caused by the selective complex is less than the skin toxicity caused by anti-EGFR antibody or tyrosine kinase inhibitor therapy, which inhibit EGFR without TfR tissue targeting. Keratinocytes may express less TfR than tumor cells. Keratinocytes can, in some instances, express 3-37-fold less TfR per cell than cancer cell lines. [0474] Cancers may also be similarly treated by using a selective depletion complex that binds both PD-Ll and EGFR.

[0475] Optionally, the cancer is treated in combination by administration of an SDC that depletes EGFR in combination with radiation, chemotherapy, platinum therapy, anti-metabolic therapy, targeted therapy to other oncogenic signaling pathways, targeted therapy to immune response pathways, therapy aimed at directly driving an immune response to cancer cells, or targeted therapies disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells.

[0476] Optionally, the patient is treated with the EGFR SDC after the patient has developed resistance to other EGFR targeted therapies, such as after treatment with EGFR antibodies like cetuximab, necitumumab, and panitumumab, or after treatment with first, second, third or other generation TKIs, such as Osimertinib, gefibitib, erlotinib, afatinib, or dacomitinib. Alternatively, the patient may be treated with the EGFR SDC as frontline therapy, which may optionally prevent or delay the time to developing resistance to EGFR-targeted therapy.

EXAMPLE 16

Treatment of Cancer by Administration of a Selective Depletion Complex [0477] This example describes treatment of cancer by administration of a selective depletion complex (SDC). An SDC containing a EGFR-b inding peptide, which is optionally pH dependent, such as that of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494, and a TfR-binding peptide, such as that of SEQ ID NO: 96, or a selective depletion complex of any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526, is administered to a subject having an EGFR positive cancer. The subject may be a human, a nonhuman primate, a mouse, a rat, or another species. A selective depletion complex may be administered subcutaneously, intravenously, intramuscularly, interperitoneally, or by another route. It may be administered 1, 2, 3, 4, 5, 10, or more times and at a frequency of 1, 2, 3, 4, 5, 6, or 7 times per week or every other week or every third week, or monthly or every other month or every third month or less frequently. The EGFR positive cancer is non-small-cell lung cancer, head and neck cancer, glioblastoma, metastatic brain cancer, primary brain cancer, EGFR VIII brain cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, TKI-resistant cancer, osimertinib-resistant cancer, gefitinib-resistant cancer, erlotinib-resistant cancer, cetuximab-resistant cancer, necitumumab-resistant cancer, or panitumumab-resistant cancer. Optionally, the cancer overexpresses EGFR, is KRAS mutant, has a KRAS G12S mutation, has a KRAS G12C mutation, has PTEN loss, has an EGFR exonl9 deletion, has an EGFR L858R mutation, has an EGFR T790M mutation, has a cetuximab-resistant EGFR, has a panitumumab- resistant EGFR, has a mutant PIK3CA, has PIK3CA G118D, has a TP53 R273H mutation, has PIK3Ca amplification, has a C79X mutation, a G724S mutation, an L718Q mutation, or an S768I mutation. The administration of the SDC causes reduced activation of oncogenic pathways in the cancer, slows cancer growth, reduces the cancer cells viability, induces senescence in the cancer cells, increases the cancer’s vulnerability to immune-mediated attack or clearance, or a combination thereof, thereby treating the patient’s cancer.

EXAMPLE 17

Treatment of KRAS-Mutant Cancer by Administration of a Selective Depletion Complex [0478] This example describes treatment of cancer by administration of an SDC. A selective depletion complex containing an EGFR-binding peptide, which is optionally pH dependent, such as that of SEQ ID NO: 318 - SEQ ID NO: 390, or SEQ ID NO: 457 - SEQ ID NO: 494, and a TfR-binding peptide, such as that of SEQ ID NO: 96, or a selective depletion complex of any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 503 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526, is administered to a subject having a KRAS-mutant, EGFR positive cancer. The subject may be a human, a non-human primate, a mouse, a rat, or another species. A selective depletion complex may be administered subcutaneously, intravenously, intramuscularly, interperitoneally, or by another route. It may be administered 1, 2, 3, 4, 5, 10, or more times and at a frequency of 1, 2, 3, 4, 5, 6, or 7 times per week or every other week or every third week, or monthly or every other month or every third month or less frequently. The KRAS-mutant, EGFR positive cancer is optionally non-small-cell lung cancer, head and neck cancer, glioblastoma, metastatic brain cancer, primary brain cancer, EGFRVIII brain cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, TKI-resistant cancer, osimertinib-resistant cancer, gefitinib-resistant cancer, erlotinib-resistant cancer, cetuximab-resistant cancer, necitumumab-resistant cancer, or panitumumab-resistant cancer. Administration of the SDC reduces pro-growth signaling in the cancer cell, slows cancer growth or metastases, or a combination thereof, thereby treating the patient’s cancer.

Administration of the SDC may cause senescence in the cancer cells, may reduce the viability of the cancer cells, and may make the cancer cells more vulnerable to immune-mediated attack and clearance.

EXAMPLE 18

Treatment of Cancer by Administration of an EGF Variant

[0479] This example describes treatment of cancer by administration of an EGF variant. EGF variant of any one of SEQ ID NO: 318 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494) or an EGF variant peptide complex (e.g., SEQ ID NO: 498 or SEQ ID NO: 502) is administered to a subject having an EGFR positive cancer. The subject may be a human, a nonhuman primate, a mouse, a rat, or another species. An EGF variant may be administered subcutaneously, intravenously, intramuscularly, interperitoneally, or by another route. It may be administered 1, 2, 3, 4, 5, 10, or more times and at a frequency of 1, 2, 3, 4, 5, 6, or 7 times per week or every other week or every third week, or monthly or every other month or every third month or less frequently. The EGFR positive cancer is non-small-cell lung cancer, head and neck cancer, glioblastoma, metastatic brain cancer, primary brain cancer, EGFRVIII brain cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, TKI-resistant cancer, osimertinib-resistant cancer, gefitinib-resistant cancer, erlotinib-resistant cancer, cetuximab-resistant cancer, necitumumab-resistant cancer, or panitumumab-resistant cancer. Optionally, the cancer overexpresses EGFR, is KRAS mutant, has a KRAS G12S mutation, has a KRAS G12C mutation, has PTEN loss, has an EGFR exonl9 deletion, has an EGFR L858R mutation, has an EGFR T790M mutation, has a cetuximab-resistant EGFR, has a panitumumab- resistant EGFR, has a mutant PIK3CA, has PIK3CA G118D, has a TP53 R273H mutation, has PIK3Ca amplification, has a C79X mutation, a G724S mutation, an L718Q mutation, or an S768I mutation. The administration of the EGV variant inhibits activation of EGFR, reduces activation of oncogenic pathways in the cancer, slows cancer growth, reduces the cancer cells viability, induces senescence in the cancer cells, increases the cancer’s vulnerability to immune- mediated attack or clearance, or a combination thereof, thereby treating the patient’s cancer. EXAMPLE 19

Delivery of a Selective Depletion Complex to the Brain

[0480] A selective depletion complex (SDC) of this disclosure is administered to a subject. The subject may be a human, a nonhuman primate, a rat, a mouse, or another mammal. The administration may be intravenous, subcutaneous, intramuscular, intrathecal, intraperitoneal, or by other route. The SDC of this disclosure binds to TfR and optionally contains a TfR-binding peptide of SEQ ID NO: 96. The SDC binds to TfR, such as that expressed on endothelial cells that are part of the blood-brain barrier. The SDC is transcytosed across the blood-brain barrier and released into the central nervous system and optionally the brain parenchyma. The SDC binds to a target in the central nervous system, such as in the brain, and depletes the target which is EGFR. The target may be cell membrane-bound or may be in solution such as in interstitial fluid. The SDC may reach the brain at concentrations sufficient to deplete the target and thereby treat a patient in need of depletion of the target. The patient may have an EGFR-expressing cancer in the brain.

EXAMPLE 20

Manufacture of a Selective Depletion Complexes

[0481] This example describes the manufacture of the SDC of SEQ ID NO: 495. Proteins of SEQ ID NO: 495 were generated in mammalian cell culture using transient transfection of plasmids driving expression of the gene encoding the SDC of SEQ ID NO: 495, further modified to carry a “signal peptide” (e.g. SEQ ID NO: 230) permitting secretion into the cell culture media, cloned using standard molecular biology techniques (M.R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). After a period of growth in suspension culture permitting accumulation of secreted protein, the media was collected. The protein of SEQ ID NO: 495 was also isolated by protein A chromatography, buffer exchanged into an appropriate buffer, and stored frozen. Analysis of the isolated protein of SEQ ID NO: 495 demonstrated good purity by SDS-PAGE (reducing and non-reducing) and by SE-HPLC.

EXAMPLE 21

Manufacture of a Selective Depletion Complexes

[0482] This example describes the manufacture of the SDCs, control complexes, and components thereof, described herein (e.g., any one of SEQ ID NO: 495 - SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526). Proteins are generated in mammalian cell culture using transient transfection of plasmids driving expression of the gene encoding the SDC, further modified to carry a “signal peptide” (e.g. SEQ ID NO: 230) permitting secretion into the cell culture media, cloned using standard molecular biology techniques (M.R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). After a period of growth in suspension culture permitting accumulation of secreted protein, the media is collected, and the proteins are isolated by immobilized metal affinity chromatography (IMAC) or by protein A chromatography, buffer exchanged into an appropriate buffer (e.g., PBS), and stored frozen.. Analysis of the isolated protein demonstrates good purity by SDS-PAGE (reducing and nonreducing) and by SE-HPLC.

EXAMPLE 22

Modifications for Improving Selective Depletion Complex Activity In Vivo [0483] This example describes modifications to SDCs that improve in vivo activity. A therapeutic selective depletion complex (SDC) incorporates domains capable of binding to a target protein to be depleted as well as to a receptor facilitating endolysosomal delivery and that can also recycle. There are many ways of altering or optimizing such a molecule for improved clinical activity. Accumulation in the target tissue, or on target cell types, can be modulated depending on binding to target protein, to uptake receptor, or to both. Accumulation in nontarget tissues or cell types can be modulated similarly. Accumulation may be improved by tighter binding to target and/or uptake receptor, or by weaker binding to target and/or uptake receptor. Tighter or weaker binding can be accomplished by altering the affinity or the avidity of a given binding domain through sequence alteration. It can also be accomplished by maintaining the same affinity and avidity but modulating the on-rate and off-rate of binding through sequence alteration.

[0484] Activity can be improved by modifying the molecule’s pharmacokinetic parameters. This can be accomplished by modifying the binding to the target (e.g., the EGF variant portion of the SDC binding to the EGFR), binding to the uptake receptor (e.g., the TfR Binder portion of the SDC biding to the TfR), and/or modifying binding to another protein. An example of the latter is SDC binding to the neonatal Fc receptor (FcRn) through incorporation and/or modification of an Fc domain in the SDC. This interaction can increase serum half-life of the SDC, subsequently increasing overall tissue exposure. Activity can also be improved by altering the protein’s stability, e.g., via alterations that improve its resistance to proteases through sequence alteration in one or more portions of the SDC. [0485] In some scenarios, a given SDC has demonstrable pharmacodynamic activity in vivo, but it’s in vivo activity is improved when its TfR-binding domains are modified from high affinity to medium affinity. This may improve in vivo activity by modulating biodistribution, permitting more SDC to reach, and permeate, the tumor after administration.

[0486] For example, while the SDC comprising SEQ ID NO: 495 (that incorporates EGF Variant SEQ ID NO: 494) has demonstrable EGFR depletion capability in cells in vitro, further modification of the sequences of the EGF variant and/or the TfR binder and/or the Fc domain and/or the linkers may improve the SDC’s pharmacodynamic activity in vivo. For the EGF variants specifically, continued engineering for stronger or weaker EGFR binding may yield a molecule with improved activity in vivo beyond what would be demonstrated with in vivo usage of the SDC comprising SEQ ID NO: 495. Similar modifications can be made for the EGF variants comprising SEQ ID NO 388 - SEQ ID NO: 390 or SEQ ID NO: 457 - SEQ ID NO: 494.

[0487] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R.

2. The EGFR-binding peptide of claim 1, wherein the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof.

3. The EGFR-binding peptide of claim 1 or claim 2, wherein the sequence further comprises one or more mutations relative to SEQ ID NO: 317 comprising DI 1R, I23S, V35E, E51P, L52E, R53E, or a combination thereof.

4. The EGFR-binding peptide of any one of claims 1-3, wherein the sequence further comprises one or more mutations relative to SEQ ID NO: 317 comprising E51H, L52H, R53H, or a combination thereof.

5. The EGFR-binding peptide of any one of claims 1-4, wherein the EGFR-binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 458 - SEQ ID NO: 493 or a fragment thereof.

6. The EGFR-binding peptide of any one of claims 1-5, wherein the sequence comprises SEQ ID NO: 494.

7. The EGFR-binding peptide of any one of claims 1-5, wherein the sequence comprises any one of SEQ ID NO: 458 - SEQ ID NO: 493.

8. The EGFR-binding peptide of any one of claims 1-7, wherein the fragment comprises at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues.

9. The EGFR-binding peptide of any one of claims 1-8, wherein the EGFR-binding peptide is capable of binding to EGFR without activating the EGFR.

10. The EGFR-binding peptide of any one of claims 1-9, wherein the EGFR-binding peptide blocks binding of EGF to EGFR when the EGFR-binding peptide is bound to the EGFR.

11. The EGFR-binding peptide of any one of claims 1-10, wherein the EGFR-binding peptide inhibits EGFR when the EGFR-binding peptide is bound to the EGFR.

12. The EGFR-binding peptide of any one of claims 1-11, wherein the EGFR-binding peptide prevents dimerization of EGFR when the EGFR-binding peptide is bound to the EGFR.

13. A peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptor-binding peptide; and the EGFR-binding peptide has affinity for a target molecule.

14. The peptide complex of claim 13, wherein the EGFR-binding peptide is the EGFR- binding peptide of any one of claims 1-11.

15. The peptide complex of claim 13 or claim 14, wherein the affinity of the EGFR-binding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for a cellular receptor, or both is pH-independent.

16. The peptide complex of any one of claims 13-15, wherein the affinity of the EGFR- binding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for the cellular receptor, or both is pH dependent.

17. The peptide complex of any one of claims 13-16, wherein the affinity of the EGFR- binding peptide for the target molecule, the affinity of the cellular receptor-binding peptide for the cellular receptor, or both is ionic strength dependent.

18. The peptide complex of any one of claims 13-17, wherein the cellular receptor-binding peptide is a transferrin receptor-binding peptide or a PD-L1 -binding peptide.

19. The peptide complex of any one of claims 13-18, wherein the cellular receptor is a transferrin receptor or PD-L1.

20. The peptide complex of any one of claims 13-19, wherein the cellular receptor is a cation-independent mannose 6 phosphate receptor (CI-M6PR), an asialoglycoprotein receptor (ASGPR), CXCR7, folate receptor, or Fc receptor (including but not limited to neonatal Fc receptor (FcRn) or FcyRIIb).

21. The peptide complex of any one of claims 13-20, wherein the cellular receptor-binding peptide binds to the cellular receptor at a pH of from pH 4.5 to pH 7.4, from pH 5.5 to pH 7.4, from pH 5.8 to pH 7.4, or from pH 6.5 to pH 7.4.

22. The peptide complex of any one of claims 13-21, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 7.4.

23. The peptide complex of any one of claims 13-22, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with an equilibrium dissociation constant (KD) of no more than 100 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM at pH 5.5.

24. The peptide complex of any one of claims 13-23, wherein the cellular receptor-binding peptide is capable of binding the cellular receptor with a dissociation rate constant (k_off or kd) of no more than 1 s'¹, no more than 5x10'¹ s'¹, no more than 2x10'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s'¹, or no more than 2x1 O'⁴ s'¹ at pH 5.5.

25. The peptide complex of any one of claims 13-24, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor is pH-independent.

26. The peptide complex of any one of claims 13-25, wherein the affinity of the EGFR- binding peptide for the target molecule is pH-dependent.

27. The peptide complex of any one of claims 13-26, wherein the affinity of the EGFR- binding peptide for the target molecule is pH-independent.

28. The peptide complex of any one of claims 13-27, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

29. The peptide complex of any one of claims 13-28, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

30. The peptide complex of any one of claims 13-29, wherein the dissociation rate constant (k_off or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.5 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15- fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

31. The peptide complex of any one of claims 13-30, wherein the dissociation rate constant (k_off or kd) of the cellular receptor-binding peptide for the cellular receptor at pH 7.4 and at pH 5.8 differs by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15- fold, no more than 20-fold, no more than 25 -fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold.

32. The peptide complex of any one of claims 13-31, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor is pH dependent.

33. The peptide complex of claim 32, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor decreases as pH decreases.

34. The peptide complex of claim 32 or claim 33, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.5.

35. The peptide complex of claim 32 or claim 33, wherein the affinity of the cellular receptor-binding peptide for the cellular receptor is higher at pH 7.4 than at pH 5.8.

36. The peptide complex of any one of claims 13-35, wherein the affinity of the EGFR- binding peptide for the target molecule is pH dependent.

37. The peptide complex of any one of claims 13-36, wherein the affinity of the EGFR- binding peptide for the target molecule decreases as pH decreases.

38. The peptide complex of any one of claims 13-37, wherein the affinity of the EGFR- binding peptide for the target molecule is higher at a higher pH than at a lower pH.

39. The peptide complex of claim 38, wherein the higher pH is pH 7.4, pH 7.2, pH 7.0, or pH 6.8.

40. The peptide complex of claim 38 or claim 39, wherein the lower pH is pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5.

41. The peptide complex of any one of claims 13-40, wherein the affinity of the EGFR- b inding peptide for the target molecule is higher at pH 7.4 than at pH 6.0.

42. The peptide complex of any one of claims 13-41, wherein the affinity of the EGFR- binding peptide for the target molecule is higher at pH 7.4 than at pH 5.5.

43. The peptide complex of any one of claims 13-42, wherein the affinity of the EGFR- b inding peptide for the target molecule is higher at pH 7.4 than at pH 5.8.

44. The peptide complex of any one of claims 13-43, wherein the EGFR-b inding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no more than 500 nM, no more than 200 nM, 100 nM, no more than 50 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 1 nM, or no more than 0.1 nM at pH 7.4.

45. The peptide complex of any one of claims 13-44, wherein the EGFR-binding peptide is capable of binding the target molecule with a dissociation rate constant (k_off or kd) of no more than 1x10'¹ s’¹, 5x1 O’² s’¹, no more than 2x1 O’² s’¹, no more than 1x1 O’² s’¹, no more than 5x1 O’³ s’¹, no more than 2x1 O’³ s’¹, no more than 1x1 O’³ s’¹, no more than 5x1 O’⁴ s’¹, no more than 2x10’ ⁴ s’¹, no more than IxlO’⁴ s’¹, no more than 5xl0’⁵ s’¹, or no more than 2xl0’⁵ s’¹ at pH 7.4.

46. The peptide complex of any one of claims 13-45, wherein the EGFR-b inding peptide is capable of binding the target molecule with a dissociation rate constant (k_off or kd) of no more than 1 s'¹, no more than 5x1 O'¹ s'¹, no more than 2x1 O'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s'¹, or no more than 2x1 O'⁴ s'¹ at pH 5.5.

47. The peptide complex of any one of claims 13-46, wherein the EGFR-binding peptide is capable of binding the target molecule with a dissociation rate constant (koff or kd) of no more than 1 s'¹, no more than 5x1 O'¹ s'¹, no more than 2x1 O'¹ s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'² s'¹, no more than 2x1 O'² s'¹, no more than 1x1 O'² s'¹, no more than 5x1 O'³ s'¹, no more than 2x1 O'³ s'¹, no more than 1x1 O'³ s'¹, no more than 5x1 O'⁴ s'¹, or no more than 2x1 O'⁴ s'¹ at pH 5.8.

48. The peptide complex of any one of claims 13-47, wherein the dissociation rate constant (koff or kd) for EGFR-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.5 than at pH 7.4.

49. The peptide complex of any one of claims 13-48, wherein the dissociation rate constant (koff or kd) for EGFR-binding peptide binding the target molecule is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 1,000 fold, at least 2,000 fold, at least 5,000 fold, at least 10,000 fold, at least 20,000 fold, or at least 50,000 fold higher at pH 5.8 than at pH 7.4.

50. The peptide complex of any one of claims 13-49, wherein the EGFR-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.5.

51. The peptide complex of any one of claims 13-50, wherein the EGFR-binding peptide is capable of binding the target molecule with an equilibrium dissociation constant (KD) of no less than 0.1 nM, no less than 0.5 nM, 1 nM, no less than 2 nM, no less than 5 nM, no less than 10 nM, no less than 20 nM, no less than 50 nM, no less than 100 nM, no less than 200 nM, or no less than 500 nM, or no less than 1000 nM at pH 5.8.

52. The peptide complex of any one of claims 13-51, wherein the affinity of the EGFR- binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.5.

53. The peptide complex of any one of claims 13-51, wherein the affinity of the EGFR- binding peptide for the target molecule at pH 7.4 is at least 1.5-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.8.

54. The peptide complex of any one of claims 13-53, wherein the affinity of the EGFR- binding peptide for the target molecule at pH 7.4 is less than 0.5-fold, less than 1-fold, less than, 1.5-fold, less than 2-fold, less than 3-fold, or less than 10-fold, greater than the affinity of the EGFR-binding peptide for the target molecule at pH 5.8.

55. The peptide complex of any one of claims 13-54, wherein the EGFR-binding peptide comprises one or more histidine amino acid residues.

56. The peptide complex of any one of claims 13-55, wherein the affinity of the EGFR- binding peptide for the target molecule decreases as ionic strength increases.

57. The peptide complex of any one of claims 13-56, wherein the EGFR-binding peptide comprises one or more polar or charged amino acid residues capable of forming polar or chargecharge interactions with the target molecule.

58. The peptide complex of any one of claims 13-57, wherein the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the EGFR-binding peptide.

59. The peptide complex of any one of claims 13-58, wherein the cellular receptor-binding peptide is fused to, linked to, complexed with, or conjugated to the EGFR-binding peptide via a polymer linker.

60. The peptide complex of claim 59, wherein the polymer linker is a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer comprising proline, alanine, serine, or a combination thereof, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, a palmitic acid, an albumin, or an albumin binding molecule.

61. The peptide complex of any one of claims 13-60 wherein the cellular receptor-binding peptide and the EGFR-binding peptide form a single polypeptide chain.

62. The peptide complex of any one of claims 13-61, wherein the peptide complex comprises a dimer dimerized via a dimerization domain.

63. The peptide complex of any one of claims 13-62, wherein a distance between the cellular receptor-binding peptide and the EGFR-binding peptide is at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, or at least 100 nm.

64. The peptide complex of claim 62, wherein the dimerization domain comprises an Fc domain.

65. The peptide complex of claim 62, wherein the dimer is a homodimer dimerized via a homodimerization domain.

66. The peptide complex of claim 65, wherein the homodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 535, SEQ ID NO: 706, or SEQ ID NO: 246.

67. The peptide complex of claim 62, wherein the dimer is a heterodimer dimerized via a first heterodimerization domain and a second heterodimerization domain.

68. The peptide complex of claim 67, wherein the first heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 536, SEQ ID NO: 707, or SEQ ID NO: 709.

69. The peptide complex of claim 67 or claim 68, wherein the second heterodimerization domain comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 537, SEQ ID NO: 708, or SEQ ID NO: 710.

70. The peptide complex of any one of claims 62-69, wherein the EGFR-binding peptide is linked to the dimerization domain via a peptide linker.

71. The peptide complex of any one of claims 62-70, wherein the cellular receptor-binding peptide is linked to the dimerization domain via a peptide linker.

72. The peptide complex of any one of claims 13-71, wherein the cellular receptor-binding peptide is linked to the EGFR-binding peptide via a peptide linker.

73. The peptide complex of claim 70-72, wherein the peptide linker has a length of from 1 to 50 amino acid residues, from 2 to 40 amino acid residues, from 3 to 20 amino acid residues, or from 3 to 10 amino acid residues.

74. The peptide complex of any one of claims 71-73, wherein the peptide linker comprises glycine and serine amino acids.

75. The peptide complex of any one of claims 71-74, wherein the peptide linker has a persistence length of no more than 6 A, no more than 8 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, no more than 50 A, no more than 75 A, no more than 100 A, no more than 150 A, no more than 200 A, no more than 250 A, or no more than 300 A.

76. The peptide complex of any one of claims 71-75, wherein the peptide linker is derived from an immunoglobulin peptide.

77. The peptide complex of any one of claims 71-75, wherein the peptide linker is derived from a double-knot toxin peptide.

78. The peptide complex of any one of claims 71-77, wherein the peptide linker comprises a sequence of any one of SEQ ID NO: 129 - SEQ ID NO: 141, SEQ ID NO: 195 - SEQ ID NO: 218, SEQ ID NO: 223 - SEQ ID NO: 223 - SEQ ID NO: 227, SEQ ID NO: 194, SEQ ID NO: 391, SEQ ID NO: 538, or SEQ ID NO: 540 - SEQ ID NO: 541.

79. The peptide complex of any one of claims 13-78, wherein the cellular receptor-binding peptide, the EGFR-binding peptide, peptide complex, or a combination thereof comprises a miniprotein, a nanobody, an antibody, an antibody fragment, an scFv, a DARPin, or an affibody.

80. The peptide complex of claim 79, wherein the antibody comprises an IgG, or wherein the antibody fragment comprises a Fab, a F(ab)2, an scFv, or an (scFv)2.

81. The peptide complex of claim 79 or claim 80, wherein the miniprotein comprises a cystine-dense peptide, an affitin, an adnectin, an avimer, a Kunitz domain, a nanofittin, a fynomer, a bicyclic peptide, a beta-hairpin, or a stapled peptide.

82. The peptide complex of any one of claims 13-81, wherein the cellular receptor-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds.

83. The peptide complex of any one of claims 13-82, wherein the EGFR-binding peptide comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds.

84. The peptide complex of any one of claims 13-83, wherein the peptide complex comprises at least one disulfide bond, at least two disulfide bonds, at least three disulfide bonds, or at least four disulfide bonds.

85. The peptide complex of any one of claims 13-84, wherein the cellular receptor-binding peptide comprises at least six cysteine residues.

86. The peptide complex of claim 85, wherein the at least six cysteine residues are positioned at amino acid positions 4, 8, 18, 32, 42, and 46 of the cellular receptor-binding peptide.

87. The peptide complex of claim 85 or claim 86, wherein the at least six cysteine residues form at least three disulfide bonds.

88. The peptide complex of any one of claims 13-87, wherein the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 148 - SEQ ID NO: 177.

89. The peptide complex of any one of claims 13-88, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of any one of SEQ ID NO: 96, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 - SEQ ID NO: 95, SEQ ID NO: 97 - SEQ ID NO: 128, SEQ ID NO: 220 - SEQ ID NO: 222, or SEQ ID NO: 1 - SEQ ID NO: 64.

90. The peptide complex of any one of claims 13-89, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 96, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 96.

91. The peptide complex of any one of claims 13-90, wherein the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 96.

92. The peptide complex of any one of claims 13-90, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 66, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 66.

93. The peptide complex of any one of claims 13-90 or claim 92, wherein the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 66.

94. The peptide complex of any one of claims 13-90, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 65, or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 65.

95. The peptide complex of any one of claims 13-90 or claim 94, wherein the cellular receptor-binding peptide comprises a sequence of SEQ ID NO: 65.

96. The peptide complex of any one of claims 13-87, wherein the cellular receptor-binding peptide comprises a sequence of any one of SEQ ID NO: 392 - SEQ ID NO: 399.

97. The peptide complex of any one of claims 13-87 or claim 96, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 187, SEQ ID NO: 233 - SEQ ID NO: 239, SEQ ID NO: 400 - SEQ ID NO: 456, or SEQ ID NO: 241.

98. The peptide complex of any one of claims 13-87, 96, or 97, wherein the cellular receptor-binding peptide comprises a sequence that has at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 187, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 400, or SEQ ID NO: 401 or at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a fragment of SEQ ID NO: 187.

99. The peptide complex of any one of claims 13-87 or 96-98, wherein the cellular receptorbinding peptide comprises a sequence of SEQ ID NO: 187.

100. The peptide complex of any one of claims 89-99, wherein the fragment comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acid residues.

101. The peptide complex of any one of claims 13-100, wherein the cellular receptor-binding peptide comprises one or more histidine residues at a cellular receptor-binding interface.

102. The peptide complex of any one of claims 13-101, wherein the EGFR-binding peptide comprises one or more histidine residues at a EGFR-binding interface.

103. The peptide complex of any one of claims 13-102, wherein the target molecule comprises an EGFR.

104. The peptide complex of claim 103, wherein the EGFR is wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, a panitumumab-resistant EGFR, or a combination thereof.

105. The peptide complex of claim 104, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

106. The peptide complex of any one of claims 13-105, wherein an off rate of the cellular receptor-binding peptide from the cellular receptor is slower than a recycling rate of the cellular receptor.

107. The peptide complex of any one of claims 13-106, wherein a half-life of dissociation of the cellular receptor-binding peptide from the cellular receptor is no faster than 1 minute, no faster than 2 minutes, no faster than 3 minutes, no faster than 4 minutes, no faster than 5 minutes, no faster than 7 minutes, no faster than 10 minutes, no faster than 15 minutes, no faster than 20 minutes, no faster than 30 minutes, no faster than 45 minutes, no faster than 60 minutes, no faster than 90 minutes, or no faster than 120 minutes.

108. The peptide complex of any one of claims 13-107, wherein a rate of dissociation of the EGFR-binding peptide from the target molecule is faster than a recycling rate of the cellular receptor.

109. The peptide complex of any one of claims 13-108, wherein a half-life of dissociation of a target binding-binding peptide from the target molecule is less than 10 seconds, less than 20 seconds, less than 30 seconds, less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, or less than 60 minutes in endosomal conditions.

110. The peptide complex of any one of claims 13-109, wherein the peptide complex is capable of being endocytosed via receptor-mediated endocytosis.

111. The peptide complex of claim 110, wherein the receptor-mediated endocytosis is transferrin receptor-mediated endocytosis.

112. The peptide complex of claim 110, wherein the receptor-mediated endocytosis is PD-L1- mediated endocytosis.

113. The peptide complex of any one of claims 13-112, wherein the cellular receptor-binding peptide remains bound to the cellular receptor inside an endocytic vesicle.

114. The peptide complex of any one of claims 13-113, wherein the peptide complex is recycled to the cell surface when the cellular receptor-binding peptide is bound to the cellular receptor and the cellular receptor is recycled.

115. The peptide complex of any one of claims 13-114, wherein the target molecule is released or dissociated from the EGFR-binding peptide after the peptide complex is endocytosed via receptor-mediated endocytosis.

116. The peptide complex of any one of claims 13-115, wherein the target molecule is an extracellular protein, a circulating protein, or a soluble protein.

117. The peptide complex of any one of claims 13-116, wherein the target molecule is a cell surface protein.

118. The peptide complex of any one of claims 13-117, wherein the target molecule is a transmembrane protein.

119. The peptide complex of any one of claims 13-118, further comprising a half-life modifying agent coupled to the cellular receptor-binding peptide, the EGFR-binding peptide, or both.

120. The peptide complex of claim 119, wherein the half-life modifying agent is a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.

121. The peptide complex of claim 120, wherein the molecule that binds to albumin is a serum albumin-binding peptide.

122. The peptide complex of claim 121, wherein the serum albumin-binding peptide comprises a sequence of any one of SEQ ID NO: 178, SEQ ID NO: 179, or SEQ ID NO: 193.

123. The peptide complex of any one of claims 13-122, wherein the cellular receptor-binding peptide, the EGFR-binding peptide, or both is recombinantly expressed.

124. The peptide complex of any one of claims 13-123, wherein the EGFR-binding peptide is configured to dissociate from the target molecule at pH 6.5, pH 6.0, pH 5.8, pH 5.5, pH 5.0, or pH 4.5.

125. The peptide complex of any one of claims 13-124, wherein the cellular receptor-binding peptide is configured to dissociate from the cellular receptor at pH 6.5, pH 6.0, pH 5.5, pH 5.0, or pH 4.5.

126. The peptide complex of any one of claims 13-125, wherein the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 499 - SEQ ID NO: 501, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 507, SEQ ID NO: 508, or SEQ ID NO: 511 - SEQ ID NO: 526.

127. The peptide complex of any one of claims 13-126, wherein the peptide complex comprises a sequence having at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 495 - SEQ ID NO: 497, SEQ ID NO: 503, or SEQ ID NO: 506.

128. A peptide-active agent complex comprising a peptide complexed to an active agent, wherein the peptide comprises the EGFR-binding peptide of any one of claims 1-12 or the peptide complex of any one of claims 13-127.

129. The peptide-active agent complex of claim 128, wherein the active agent comprises a peptide, a peptidomimetic, an oligonucleotide, a DNA, an RNA, an antibody, a single chain variable fragment (scFv), an antibody fragment, an aptamer, or a small molecule.

130. The peptide-active agent complex of claim 129, wherein the DNA comprises cDNA, ssDNA, or dsDNA.

131. The peptide-active agent complex of claim 129, wherein the RNA comprises RNAi, microRNA, snRNA, dsRNA, or an antisense oligonucleotide.

132. The peptide-active agent complex of any one of claims 128-131, wherein the active agent is a therapeutic agent or a detectable agent.

133. The peptide-active agent complex of claim 132, wherein the detectable agent comprises a dye, a fluorophore, a fluorescent biotin compound, a luminescent compound, a chemiluminescent compound, a radioisotope, nanoparticle, a paramagnetic metal ion, or a combination thereof.

134. The peptide-active agent complex of claim 132, wherein the therapeutic agent comprises a chemical agent, a small molecule, a therapeutic, a drug, a peptide, an antibody protein, any fragment thereof, or any combination thereof.

135. The peptide-active agent complex of any one of claims 132-134, wherein the therapeutic agent comprises an oncology agent, an autoimmune disease agent, an acute and chronic neurodegeneration agent, a pain management agent, or an anti-cancer agent.

136. The peptide-active agent complex of claim 135, wherein the anti-cancer agent comprises a radionuclide, radioisotope, a chemotherapeutic agent, a platinum therapeutic, a toxin, an enzyme, a sensitizing drug, an anti-angiogenic agent, cisplatin, an anti-metabolite, an anti- metabolic therapeutic, a mitotic inhibitor, a growth factor inhibitor, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, or amifostine.

137. The peptide-active agent complex of claim 135 or claim 136, wherein the anti-cancer agent targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells.

138. A pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent.

139. The pharmaceutical composition of claim 138, wherein the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof.

140. The pharmaceutical composition of claim 138, wherein the EGFR-binding peptide is the EGFR-binding peptide of any one of claims 1-12.

141. A pharmaceutical composition comprising: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent.

142. The pharmaceutical composition of claim 141, wherein the peptide complex is the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

143. A method of administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent, to a subject.

144. The method of claim 143, wherein the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof.

145. The method of claim 146, wherein the EGFR-binding peptide is the EGFR-binding peptide of any one of claims 1-12.

46. A method of administering a pharmaceutical composition comprising: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent, a subject.

147. The method of claim 146, wherein the peptide complex is the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

148. The method of any one of claims 143-147, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 138-142.

149. A method of inhibiting EGFR in a subject, the method comprising: administering to the subject a pharmaceutical composition, wherein the pharmaceutical composition comprises: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR.

150. The method of claim 149, wherein the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof.

151. The method of claim 149, wherein the EGFR-binding peptide is the EGFR-binding peptide of any one of claims 1-12.

152. A method of inhibiting EGFR in a subject, the method comprising: administering to the subject a pharmaceutical composition, wherein the pharmaceutical composition comprises: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, 138D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide binds to EGFR on the cell of the subject and inhibits activation of the EGFR.

153. The method of claim 152, wherein the peptide complex is the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

154. The method of any one of claims 149-153, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 142-145.

155. The method of any one of claims 149-154, wherein the EGFR-binding peptide inhibits activation of the EGFR by disrupting multimerization, dimerization, or heterodimerization of the EGFR on the cell of the subject that expresses EGFR.

156. A method of selectively depleting a target molecule, the method comprising:

(a) contacting a peptide complex to a cell expressing a cellular receptor, wherein the peptide complex comprises:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, 138D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule;

(b) binding the EGFR-binding peptide to the target molecule under extracellular conditions;

(c) binding the cellular receptor-binding peptide to the cellular receptor under extracellular conditions; and

(d) endocytosing the peptide complex, the target molecule, and the cellular receptor into an endocytic or lysosomal compartment, thereby depleting the target molecule.

157. The method of claim 156, wherein the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

158. The method of claim 156 or claim 157, further comprising: (e) dissociating the EGFR-binding peptide from the target molecule, the cellular- receptor-binding peptide from the cellular receptor, or both, under endosomal or lysosomal conditions.

159. The method of any one of claims 156-158, further comprising:

(f) degrading the target molecule, thereby further depleting the target molecule.

160. The method of any one of claims 156-159, further comprising recycling the peptide complex and the cellular receptor to the cell surface.

161. A method of treating a disease or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent, to the subject, thereby treating the disease or condition.

162. A method of treating a disease or condition in a subject in need thereof, the method comprising: administering a pharmaceutical composition comprising: an EGFR-binding peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-binding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-binding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition.

163. The method of claim 161 or claim 162, wherein the EGFR-binding peptide comprises a sequence having at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 494 or a fragment thereof.

164. The method of claim 161 or claim 162, wherein the EGFR-binding peptide is the EGFR- binding peptide of any one of claims 1-12.

165. A method of treating a disease or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and (ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent, to the subject, thereby treating the disease or condition.

166. A method of treating a disease or condition in a subject in need thereof, the method comprising: administering a pharmaceutical composition comprising: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, 138D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent; and delivering the EGFR-b inding peptide to a cell of the subject that expresses EGFR, wherein the EGFR-b inding peptide inhibits the EGFR on the cell of the subject, thereby treating the disease or condition.

167. The method of claim 165 or claim 166, wherein the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

168. The method of any one of claims 161-167, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 138-142.

169. The method of any one of claims 161-168, wherein the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximabresistant EGFR, or a panitumumab-resistant EGFR.

170. The method of claim 169, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

171. A method of treating a disease or condition in a subject in need thereof, the method comprising:

(a) administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises: a peptide complex comprising:

(i) a cellular receptor-binding peptide; and

(ii) an EGFR-binding peptide, wherein: the EGFR-binding peptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 494 or a fragment thereof, wherein the sequence comprises: a first cysteine amino acid residue, a second cysteine amino acid residue, a third cysteine amino acid residue, a fourth cysteine amino acid residue, a fifth cysteine amino acid residue, and a sixth cysteine amino acid residue, wherein: there are seven amino acid residues between the first cysteine amino acid residue and the second cysteine amino acid residue, there are five amino acid residues between the second cysteine amino acid residue and the third cysteine amino acid residue, there are ten amino acid residues between the third cysteine amino acid residue and the fourth cysteine amino acid residue, there is one amino acid residue between the fourth cysteine amino acid residue and the fifth cysteine amino acid residue, and there are eight amino acid residues between the fifth cysteine amino acid residue and the sixth cysteine amino acid residue; and at least six mutations relative to SEQ ID NO: 317 comprising amino acid substitutions of M21R, A30W, V34S, I38D, K48T, and W49R; the EGFR-binding peptide is complexed with the cellular receptorbinding peptide; and the EGFR-binding peptide has affinity for a target molecule; and a pharmaceutically acceptable excipient or diluent;

(b) binding the EGFR-binding peptide under extracellular conditions to a target molecule associated with the disease or condition on a cell of the subject expressing the target molecule and a cellular receptor;

(c) binding the cellular receptor-binding peptide under extracellular conditions to the cellular receptor on the cell of the subject; and

(d) endocytosing the peptide complex, the target molecule, and the cellular receptor, thereby treating the disease or condition.

172. The method of claim 171, wherein the peptide complex is the peptide complex of any one of claims 13-127 or the peptide-active agent complex of any one of claims 128-137.

173. The method of claim 171 or claim 172, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 138-142.

174. The method of any one of claims 171-173, further comprising:

(e) dissociating the EGFR-binding peptide from the target molecule, the cellular- receptor-binding peptide from the cellular receptor, or both under endosomal conditions.

175. The method of any one of claims 171-174, further comprising:

(f) degrading the target molecule.

176. The method of any one of claims 165-175, wherein the target molecule comprises an EGFR.

177. The method of claim 176, wherein the EGFR comprises wild-type EGFR, EGFRvIII, tyrosine kinase inhibitor-resistant EGFR, EGFR containing an exon 19 deletion, EGFR containing an exon21 L858R mutation, EGFR mutant T790M, a cetuximab-resistant EGFR, or a panitumumab-resistant EGFR.

178. The method of claim 177, wherein the tyrosine kinase inhibitor-resistant EGFR comprises a EGFR L692V mutant, EGFR E709K mutant, EGFR L718Q mutant, EGFR L718V mutant, EGFR G719A mutant, EGFR G724S mutant, EGFR L747S mutant, EGFR D761Y mutant, EGFR S768I mutant, EGFR SV768IL mutant, EGFR G769X mutant, EGFR T790M mutant, EGFR L792X mutant, EGFR G796R mutant, EGFR G796S mutant, EGFR G796D mutant, EGFR C797X mutant, EGFR L798I mutant, EGFR V834I mutant, EGFR V834L mutant, EGFR V843I mutant, EGFR T854I mutant, or EGFR H870R mutant.

179. The method of any one of claims 161-178, wherein the disease or condition is a cancer.

180. The method of claim 179, wherein the cancer expresses EGFR, overexpresses EGFR, or contains mutant EGFR.

181. The method of claim 179 or claim 180, wherein the cancer is breast cancer, liver cancer, colon cancer, brain cancer, leukemia, lymphoma, non-Hodgkin lymphoma, myeloma, blood- cell-derived cancer, lung cancer, sarcoma, stomach cancer, a gastrointestinal cancer, glioblastoma, head and neck cancer, squamous head and neck cancer, non-small-cell lung cancer, squamous non-small cell lung cancer, pancreatic cancer, ovarian cancer, endometrial cancer, blood cancer, skin cancer, liver cancer, kidney cancer, or colorectal cancer.

182. The method of any one of claims 179-181, wherein the cancer is TKI -resistant, cetuximab-resistant, necitumumab-resistant, or panitumumab-resistant.

183. The method of any one of claims 179-182, wherein the cancer has one or more of the following: overexpresses EGFR, KRAS mutation, KRAS G12S mutation, KRAS G12C mutation, PTEN loss, EGFR exonl9 deletion, EGFR L858R mutation, EGFR T790M mutation, PIK3CA mutation, TP53 R273H mutation, PIK3CA amplification, PIK3CA G118D, TP53 R273H, EGFR C797X mutation, EGFR G724S mutation, EGFR L718Q mutation, EGFR S768I mutation, an EGFR mutation, a cetuximab-resistant EGFR, a panitumumab-resistant EGFR, or a combination thereof.

184. The method of claim any one of claims 179-183, wherein the cancer expresses or has upregulated c-MET, Her2, Her3 that heterodimerizes with EGFR.

185. The method of any one of claims 179-184, wherein the cancer is a primary cancer, an advanced cancer, a metastatic cancer, a metastatic cancer in the central nervous system, a primary cancer in the central nervous system, metastatic colorectal cancer, metastatic head and neck cancer, metastatic non-small-cell lung cancer, metastatic breast cancer, metastatic skin cancer, a refractory cancer, a KRAS wild type cancer, a KRAS mutant cancer, or an exon20 mutant non-small-cell lung cancer.

186. The method of any one of claims 161-185, further comprising administering an additional therapy to the subject.

187. The method of claim 186, where the additional therapy is adjuvant, first-line, or combination therapy.

188. The method of claim 186 or claim 187, wherein the additional therapy targets other oncogenic signaling pathways, targets immune response pathways, directly drives an immune response to cancer cells, or targets disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells.

189. The method of claim 188, wherein targeting of other oncogenic signaling pathways comprises administration of inhibitors of MEK/ERK pathway signaling, PI3K/AKT pathway signaling, JAK/STAT pathway signaling, or WNT/p-catenin pathway signaling.

190. The method of claim 188, wherein targeting of immune response pathways comprises PD-1/PD-L1 checkpoint inhibition.

191. The method of claim 188, wherein directly driving an immune response to cancer cells comprises bispecific T cell engagers or chimeric antigen receptor expressing T cells.

192. The method of claim 188, wherein the targeting disrupting the growth, metabolism, or oncogenic signaling capabilities of senescent cells comprises administering senolytic agents to a subject.

193. The method of any one of claims 186-192, wherein the additional therapy comprises administering fluorouracil, FOLFIRI, irinotecan, FOLFOX, gemcitabine, or cisplatin, irinotecan, oxiplatin, fluoropyrimidine to the subject.

194. The method of any one of claims 146-148, 152-160, 165-193, further comprising forming a ternary complex between the peptide complex, the target molecule, and the cellular receptor.

195. The method of claim 194, wherein formation of the ternary complex increases, facilitates, or stabilizes recycling or turnover of the cellular receptor, the target molecule, or both.

196. The method of claim 194 or claim 195, wherein formation of the ternary complex increases, facilitates, or stabilizes binding of the target molecule to the cellular receptor.

197. The method of any one of claims 146-148, 152-160, 165-196, wherein the peptide complex binds at higher levels to cells that overexpress the target molecule and the cellular receptor than to cells that have lower levels of the target molecule or the cellular receptor or both.

198. The method of any one of claims 146-148, 152-160, 165-197, wherein the peptide complex has a larger, longer, or wider therapeutic window as compared to an alternative therapy.

199. The method of claim 198, wherein the alternative therapy is not recycled to the cell surface.

200. The method of claim 198 or claim 199, wherein the alternative therapy is a lysosomal targeting therapy, a ubiquitin-proteosome system (UPS) targeting therapy, a non-selective therapeutic agent, an existing biologic, or a lysosomal delivery molecule.

201. The method of any one of claims 143-200, wherein the peptide complex or the EGFR- binding peptide is administered at lower molar dosage than alternative therapies.

202. The method of claim 143-201, wherein the peptide complex or the EGFR-binding peptide binds at higher levels to cancer cells than to normal cells.

203. The method of claim 143-202, wherein the peptide complex or the EGFR-binding peptide has a higher antiproliferative effect, a higher target molecule depletion effect, or a higher viability effect on cancer cells than on normal cells in vitro or in vivo.

204. The method of claim 143-203, wherein the peptide complex or the EGFR-binding peptide has a larger, longer, or wider therapeutic window than an anti-EGFR antibody or a TKI.

205. The method of claim 143-204, wherein the peptide complex or the EGFR-binding peptide has lower toxicity on skin or on keratinocytes than an anti-EGFR antibody or a TKI.

206. The method of any one of claims 161-205, further comprising causing remission in, reducing, ameliorating, or ablating the disease or condition.

207. The method of any one of claims 179-206, further comprising causing remission in, reducing, ameliorating, or ablating the cancer.