WO2025184423A1

WO2025184423A1 - Cell-surface-targeting-domain-polymer-payload conjugate

Info

Publication number: WO2025184423A1
Application number: PCT/US2025/017717
Authority: WO
Inventors: Lili Liu; Hong Liang; Daniel Victor Perlroth; Fernando CORRÊA
Original assignee: Kodiak Sciences Inc
Current assignee: Kodiak Sciences Inc
Priority date: 2024-02-29
Filing date: 2025-02-27
Publication date: 2025-09-04
Anticipated expiration: 2026-08-29

Abstract

Provided herein are cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) capable of delivering and internalizing high payload concentrations to targeted cells through high payload-to-cell-surface-targeting-domain ratios and/or specific and high affinity interactions between CSTDPPCs and cell surface targets. Provided herein are also methods of using CSTDPPCs to deliver payloads to targeted cells.

Description

CELL-SURFACE-TARGETING-DOMAIN-POLYMER-PAYLOAD CONJUGATE

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. This Application claims priority to U.S. Provisional Application No. 63/559742, filed February 29, 2024. The entire contents of this related application are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SequenceListingKDIAK225WO.xml created on February 27, 2025, which is 3,475 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

[0003] The present invention relates to constructs and methods thereof for delivering payloads to the intracellular space of cells.

BACKGROUND

[0004] Antibody-drug conjugates (ADCs) have been used to treat diseases, including cancer, by delivering a drug (payload) to cells that express the target antigen. Once bound to the target on the cell surface, ADC can be internalized, which permits the drug to exert its cytotoxic or therapeutic effect on the cell.

SUMMARY

[0005] Provided herein is a method for delivering a payload to a cell, the method comprising providing a cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC), wherein the cell-surface-targeting domain (CSTD) is configured for binding to the target on the surface of the cell, wherein the polymer is a copolymer (e.g., a random copolymer) and wherein the copolymer comprises one or more polymer arms extending from an initiator fragment, each polymer arm comprises a first monomer and a second monomer, wherein the first monomer further comprises a zwitterionic moiety, and the second monomer, optionally wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400; and administering the CSTDPPC to a cell, wherein binding results in internalization thereby internalizing the one or more payloads, and wherein the internalized one or more payloads is/are released from the polymer. In some embodiments, the first and second monomers are distributed randomly throughout the copolymer. In some embodiments, each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone. In some embodiments, the one or more payloads are linked to the polymer via the second monomer. In some embodiments, the copolymer comprises multiple polymer arms extending from the initiator fragment. In some embodiments, the initiator fragment is a branched initiator fragment.

[0006] Provided herein is cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPC) comprising a cell-surface-targeting domain; a polymer conjugated to the cell-surface-targeting-domain, wherein the polymer is a copolymer (e.g., a random copolymer) and wherein the copolymer comprises one or more polymer arms extending from an initiator fragment, each polymer arm comprises a first monomer and a second monomer, wherein the first monomer further comprises a zwitterionic moiety, and the second monomer; and one or more payloads linked to the copolymer and/or the CSTD, optionally wherein the one or more payloads-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400, and wherein the cell-surface-targeting domain is a cell-surface-targeting domain that is internalized upon binding to the target on a cell surface so as to allow for the CSTDPPC to be internalized thereby internalizing the one or more payloads. In some embodiments, the first and second monomers are distributed randomly throughout the copolymer. In some embodiments, each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone. In some embodiments, the one or more payloads are linked to the polymer via the second monomer. In some embodiments, the copolymer comprises multiple polymer arms extending from the initiator fragment. In some embodiments, the initiator fragment is a branched initiator fragment.

[0007] Provided herein is a cell-surface-targeting-domain-polymer-fluorescent dye conjugate (CSTD) comprising a CSTD comprising an antibody (for example, but not limited to an anti-VEGFR2 antibody), a polymer conjugated to the antibody; and a fluorescent dye, wherein the fluorescent dye is covalently linked to the antibody and/or the polymer. In some embodiments, the polymer is a homopolymer. In some embodiments, the polymer is a copolymer. In some embodiments, the fluorescent dye is covalently linked to the antibody. In some embodiments, the fluorescent dye is covalently linked to the polymer.

[0008] Provided herein is a method for delivering a payload to a cell, the method comprising providing a cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC); and administering the CSTDPPC to a cell, wherein the polymer is a copolymer (e.g., a random copolymer) comprising multiple polymer arms extending from a branched initiator fragment, each polymer arm comprises a first monomer and a second monomer distributed randomly throughout the copolymer, each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone, wherein the first monomer further comprises a zwitterionic moiety, and the second monomer is linked to the payload, optionally wherein the payload-to-cell-surface-targeting domain (“PC STD”) ratio is between 1-400, wherein the cell-surface-targeting-domain (CSTD) binds to one or more targets on the surface of a cell, wherein when the CSTD is bound to the target on the surface of the cell, the payload is released from the polymer, and wherein the payload drives internalization into the cell thereby internalizing the payload.

[0009] Provided herein is a cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising a cell-surface-targeting-domain; a polymer conjugated to the cell-surface-targeting-domain; wherein the polymer is a copolymer (e.g., a random copolymer) comprising multiple polymer arms extending from a branched initiator fragment, each polymer arm comprises a first monomer and a second monomer distributed randomly throughout the copolymer, each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone, wherein the first monomer further comprises a zwitterionic moiety, and the second monomer is linked to the payload; and a payload covalently linked to the polymer and/or the CSTD, optionally wherein the payload- to-cell-surface-targeting domain (“PC STD”) ratio is between 1-400, wherein the payload is conjugated to a peptide, wherein the peptide is linked to the polymer by a cleavable linker, and wherein the peptide drives cell internalization of the payload upon cleavage.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 depicts a schematic diagram of some embodiments of a cell-surface- targeting-domain-polymer-payload conjugate (CSTDPPC) 100.

[0011] FIG. 2A is a graph of an ELISA assay comparing VEGF-binding of an anti- VEGF antibody to its biopolymer conjugate (anti-VEGF ABC) using a VEGF-coated streptavidin plate.

[0012] FIG. 2B is a graph of an ELISA assay comparing VEGF-binding of an anti- VEGF antibody to its biopolymer conjugate (anti-VEGF ABC) using a VEGF-coated Maxisorp plate.

[0013] FIG. 2C is a graph of an in-solution binding ELISA assay comparing VEGF-binding of an anti-VEGF antibody to its biopolymer conjugate (anti-VEGF ABC) in solution.

[0014] FIG. 3 is a graph of an ELISA assay characterizing the potency of the purified anti-VEGFR2 antibody.

[0015] FIG. 4 is a graph showing the fluorescence signals of the antibodies and biopolymer conjugated antibodies at different pH conditions.

[0016] FIG. 5A is a graph showing the potency of the antibodies, biopolymer conjugated antibodies, and fluorescent-dye-conjugated antibodies and biopolymer conjugated antibodies in inhibiting VEGF-induced VEGFR2/KDR signaling in KDR/NFAT-293 cells.

[0017] FIG. 5B is a graph of an in-solution binding ELISA assay comparing VEGF-binding of the antibodies, biopolymer conjugated antibodies, and fluorescent-dye- conjugated antibodies and biopolymer conjugated antibodies. [0018] FIG. 6 depicts a schematic diagram of some embodiments of Cell-Surface- Targeting-Domain-Polymer-Fluorescent Dye-Conjugate (CSTDPFDC) 101 bound to a target on the surface of a cell 501.

[0019] FIG. 7 is a graph showing the internalized fluorescent signal in cells treated with fluorescent-dye-conjugated antibody-biopolymer conjugates and fluorescent-dye conjugated antibodies.

[0020] FIG. 8 shows images of the internalized fluorescent signal in cells incubated with fluorescent dye-conjugated antibodies and fluorescent dye-conjugated- antibody-biopolymer-conjugates.

[0021] FIG. 9 shows some embodiments of the amino acid sequence of a VEGF antibody, according to some non-limiting embodiments.

[0022] FIG. 10 is a graph showing size-exclusion chromatography with multiangle light scattering (SEC-MALS) characterization of anti -transferrin receptor 1 (TfRl)- antibody biopolymer conjugate-oligonucleotide (ABCO)-antisense oligonucleotides (ASOs).

[0023] FIG. 11 shows a graph depicting some embodiments of the kinetics of enzymatic-mediated siRNA release.

[0024] FIGS. 12A - 12D show graphs of Biacore™ surface plasmon resonance (SPR) analysis of TfRl binding to anti-TfRl antibody (FIG. 12A) and its conjugates, ABC1 (FIG. 12B), ABC2 (FIG. 12C), and ABCO1 (FIG. 12D) captured on a series S sensor protein A Chip, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.

[0025] FIGS. 13A -13E show graphs of Biacore™ SPR analysis of anti-TfRl antibody (FIG. 13A), and its conjugates, ABC1 (FIG. 13B), AOC1 (FIG. 13C), ABCO1 (FIG. 13D) and ABCO2 (FIG. 13E) binding to TfRl which was immobilized on series S sensor CM5 chips surface, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.

[0026] FIGS. 14A -14C show graphs of Biacore™ SPR analysis of anti-TfRl antibody (FIG. 14A), and its conjugates, ABC1 (FIG. 14B), and ABCO3 (FIG. 14C), binding to TfRls which was immobilized on series S sensor CM5 chips surface, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black. [0027] FIG. 15 shows a bar graph measuring the internalization of anti-TfRl - ABCO-siRNAs in HepG2 cells. The cells were treated with low pH fluorescent dye (pHAb) labeled anti-TfRl antibody, ABC1, ABCO1 and negative control antibody (NC-mAb), each at a concentration of 6.3 nM, 25 nM, or 100 nM.

[0028] FIGS. 16A and 16B show bar graphs measuring in vitro PCSK9 gene knockdown in HepG2 cells. FIG. 16A shows the relative PCSK9 mRNA level in HepG2 cells three days after reverse transfection with either free siRNA or siRNA conjugated on copolymer at 1 nM. FIG. 16B shows the relative PCSK9 mRNA level in HepG2 cells, which were treated with ABCO1 or ABC1 at 45 nM for three days followed by switching to complete growth media for an additional two days.

[0029] FIGS. 17A and 17B show line graphs measuring the relative PCSK9 mRNA level in HepG2 cells, which were treated with ABCO3 across a serial titration range from 62.5 nM to 0.004 nM for three days followed by switching to complete growth media for additional two days. FIG. 17A is a graph plotted against ABCO concentrations. FIG. 17B is a graph plotted against siRNA concentrations.

[0030] FIG. 18A shows a graph measuring internalization of anti-TfRl ABCOs with different DARs of ASO in HepG2 cells.

[0031] FIG. 18B shows a graph measuring internalization of anti-TfRl ABCOs with different sizes of copolymers in HepG2 cells.

[0032] FIG. 19 shows a graph measuring the PCSK9 gene knockdown by anti- TfRl ABCOs with different DARs of ASO in HepG2 cells.

[0033] FIGS. 20A -20B show graphs of Biacore™ SPR analysis of anti-TfRl aptamer (FIG. 20A) and its conjugate ABC5 (FIG. 20B) binding to TfRl protein captured on series S sensor protein A Chip, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.

[0034] FIGS. 21A and 21B show graphs of Biacore™ SPR analysis of anti-TfRl aptamer (FIG. 21A) and its conjugate ABC6 (FIG. 21B) binding to TfRl protein captured on series S sensor protein A Chip, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black. [0035] FIG. 22A shows a graph measuring the internalization of pHrodo deep red labeled anti-TfRl aptamer and its conjugate ABC5 in HepG2 cells.

[0036] FIG. 22B shows a graph measuring the internalization of pHrodo deep red labeled anti-TfRl aptamer and its conjugate ABC6 in HepG2 cells.

[0037] FIG. 23A shows a graph measuring the SEC-UV of Antibody Biopolymer Conjugate Drug (ABCD)-monomethyl auristatin E (MMAE) with and without 24-hour incubation with Cathepsin B.

[0038] FIG. 23B shows a graph measuring the SEC-UV of ABCD-PEG4-MMAE with and without 24-hour incubation with Cathepsin B.

[0039] FIG. 23C shows a graph measuring the SEC-UV of ABCD-PEG4-MMAF with and without 24-hour incubation with Cathepsin B.

[0040] FIGS. 24A and 24B show graphs measuring internalization of pHAb labeled anti-HER2 antibody and its conjugate ABC4 in SKBR3 cells (FIG. 24A) and MCF7 cells (FIG. 24B).

[0041] FIGS. 25A -25B show graphs measuring the cell viability of SKBR3 cells (A) and MCF7 cells (B) after three days of treatment with anti-HER2 antibody, ABC4, ABCD1, ABCD2 or ABCD3.

[0042] FIG. 26 shows OG1802.

DETAILED DESCRIPTION

[0043] The use of antibody drug conjugates (ADCs) to treat diseases, including cancer, has markedly expanded, with more than 10 ADCs approved by the United States Food and Drug Administration (FDA) and over 100 ADCs in clinical development worldwide. ADCs follow the principles of a “magic bullet”, a term coined by the Nobel laureate Paul Ehrlich (1854-1915) to describe a therapeutic agent designed to reach the cellular target while being harmless to healthy cells and tissues. The molecule design relies on the covalent linkage of a cytotoxic drug (payload) via stable or degradable linker to a monoclonal antibody (mAb) with target specificity to cells expressing its antigen. Once delivered in the body, ADC primary action is to bind to an antigen on the surface of the targeted cell, an event that is followed by potential internalization of the ADC. Breakdown of the linker between the payload and the antibody intracellularly and/or extracellularly promotes release of the payload where it exerts cytotoxic function. [0044] Major advancements have been done on the field of ADCs including: i) introduction of humanized mAbs for improved safety and efficacy; ii) design of more potent payloads with improved water solubility and coupling efficiency; iii) development of linkers with improved plasma stability and more controllable release, therefore providing less off- target toxicity; and iv) generation of more homogenous ADCs with the introduction of sitespecific conjugation approaches for better defined payload amounts and less impact on the antibody quality attributes (e.g. aggregation).

[0045] Nonetheless, ADCs have yet to reach their full potential as therapeutics. The drug-to-antibody ratio (DAR) is an important determinant of ADC efficacy. The drug-to- antibody ratio (DAR), a critical ADC attribute for drug efficacy, still falls behind. DAR is defined as the average number of drug molecules conjugated to a single monoclonal antibody (mAb). Because drugs are covalently linked to the antibody, ADC DARs are limited by factors such as the number of conjugatable sites on the antibody and the detrimental effects of heavily modified antibodies on toxicity and pharmacokinetics. A low DAR may limit the potency of the therapeutic, while a high DAR may impact ADC structure, stability, target binding, in addition to hindering other pharmacokinetic properties due to increased hydrophobicity and reduced solubility. Higher DAR has been associated with increased potency in vitro (Catcott et al. 2016) (Hamblett et al. 2004), nonetheless most ADCs on the market or in clinical development have DAR within a range between 2 and 8 ((Abuhelwa et al. 2022)(Fu et al. 2022)). The combined limited number of disponible reactive sites for conjugation (e g. number of lysine and cysteines in sequence) with the detrimental effects of heavily modifying mAbs (toxicity and poor pharmacokinetics) have hampered the generation of ADCs with higher DAR.

[0046] An antibody-oligonucleotide conjugate (AOC) is a newer class of biopharmaceuticals when compared to ADCs. Like ADCs, the three main components of an AOC are the mAb, an oligonucleotide payload and a chemical linker. The payload exploits the natural intracellular defense mechanism of RNA interference (RNAi) to mediate biological responses through degradation of targeted mRNAs. RNAi-based approaches have innate advantages over small molecules and mAbs because they execute function by Watson-Crick base pairing with mRNA, while the other two rely on complex spatial interaction with target molecules. In theory, any gene of interest could be targeted by RNAi, therefore it has potential to treat a myriad of diseases including ones with previously undruggable targets. Nonetheless, clinical success is low, and drugs have been limited to treatment of liver diseases with oligonucleotide loaded lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates.

[0047] Oligonucleotide-based therapies face challenges with bioavailability inherent from issues with stability, tissue specificity, cellular uptake, and endosomal escape. Stability issues have been largely addressed with the incorporation of chemical modifications such as phosphate backbone modifications (e.g. phosphorothioate) to impart endonuclease resistance, and ribose modifications at the 2’0 position of RNA and 2’0 position of DNA, with 2’-O-methyl, 2’0-methoxy-ethyl and 2’-fluoro improving binding affinity to RNA and enhancing nuclease resistance. Payload delivery is the dominant issue, though various approaches derived from lipids, polymers and peptides are available. Amongst these, AOCs are an ideal mechanism for delivering genetic payloads given the broader target capability for extrahepatic delivery, and ability to transport payloads across the cell membrane to the cytoplasm through antibody receptor-mediated endocytosis. There are at least 20 AOCs in pre- clinical and clinical development (Mullard 2021)(Holz et al. 2023). However, AOC DAR range is low, 1-4 (majority 1-2), which can be attributed to the challenges of directly attaching these negatively charged molecules to the antibodies without impacting their native conformation and binding capabilities. Approaches to increase AOC DAR have been tried, but product viability is still to be demonstrated. These include an AOC with a DAR of 6 based on Fab protamine conjugates (Song et al. 2005), and an AOC with a DAR of 24 based on antibody-spherical nucleic acid conjugates (Aarela et al. 2023).

[0048] The conjugation of payloads to hydrophilic polymers on antibody biopolymer conjugates offers an elegant solution to ADC and AOC DAR limitations allowing for high drug loading without compromising conjugated-mAb attributes. ADCs conjugated to poly- 1 -hydroxymethylethylene polymers are currently in clinical development and have shown some success in increasing DARs to about 20 (Yurkovetskiy et al. 2015). Random copolymers of hydrophilic polyethylene glycol (PEG)-containing monomers and hydrophobic drug-containing have also shown some success in increasing ADC DAR up to 34.5 (Kanjilal et al. 2023).

[0049] Hydrophilic polymer conjugates for drug delivery have been well described. PEG, as described above, is one example of a hydrophilic polymer, but there are several others including but not limited to poly(2-methaciyloyloxyethyl phosphorylcholine) (PMPC), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylenimine (PEI). PMPC is a biocompatible polymer widely used in a diverse range of applications in medicine such as coronary drug eluting stents, contact lenses and half-life extending conjugate in the context of biological therapeutics. The zwitterionic phosphorylcholine (PC) group composition provides unique hydration properties to this type of polymer when compared to other water-soluble polymers such as PEG. This special character contributes to PMPC high hydrophilicity and high resistance to nonspecific protein adsorption, cell adhesion, blood coagulation and cell membrane penetration. Not to mention, the PC polar head is a major component of the phospholipids present on the outer layer of the cell membrane, which grants distinctive biomimetic properties and superior biocompatibility to this type of polymer.

[0050] Antibody biopolymer platforms based on the site-specific attachment of multibranched high molecular weight (HMW) PMPC polymers to mAbs (US11584790B2, US11066465B2, 2019/0270806, US8846021B2, US8765432B2, US11819531B2,

US9840553B2, US9840553B2, and US11155610B2) have generated drug conjugates with increased half-life and intact bioactivity. Introduction of functionalized comonomers into linear or multibranched versions of these HMW PMPC polymers to generate MPC-based copolymers allows for the clicking of variable payload amounts defined by the chosen number of reactive sites (e.g. 5%, 10%, 25%). Conjugation of such copolymers to an antibody result in ADCs and AOCs with diverse engineerable DAR values (low to high) dependent on the copolymer design.

[0051] Depending on the ADC and AOC design, intracellular delivery is a key step for the drug biological response. Interactions between antibody and cell surface receptors can induce a receptor-mediated endocytosis response that will result on the internalization of antibody and conjugated payload. Downstream processing within the endosomal-lysosomal system results on delivery of payload to the cytosol. The internalization of antibody-polymer loaded molecules is a promising route to effectively increase the amounts of delivered drugs inside the cell.

[0052] Provided herein are cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) capable of delivering high concentrations of payloads to the internal spaces of targeted cells through high payload-to-cell -surface-targeting domain (“PCSTD”) ratios achieved through conjugation of payloads to the polymers and/or specific and high affinity interactions between CSTDPPCs and cell surface targets (CSTs). In some embodiments, the PCSTD ratio is more than 1, for example 5, 10, or 20 or more to 400. Provided herein are also methods of using CSTDPPCs to deliver and internalize payloads into the internal spaces of targeted cells. In some embodiments, the CSTDPPCs and methods can be used to deliver and internalize payloads into cells of a subject.

[0053] In some embodiments, large conjugates comprising an antibody, a polymer, and a payload can be internalized into cells thereby internalizing some or all of the conjugate including a payload associated with the conjugate (a CSTDPPC). This was unexpected given the size of such large molecules and allows for further molecules with therapeutic properties (i.e., payloads) to be delivered to the inside of a cell where they can have a biological effect.

[0054] In some embodiments, the cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprises at least one cell-surface targeting domain (CSTD), a polymer, and at least one payload. In some embodiments, the CSTDPPC can be targeted to cells through an interaction between the CSTD and a cell-surface target (CST). In some embodiments, delivery of the CSTDPPC to cells and the interaction between the CSTD and CST results in internalization of the payload.

TERMS

[0055] A “cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC)” as used herein refers to a modular construct comprising at least one cell-surface targeting domain, at least one polymer, and at least one payload that can be targeted to select cells. In some embodiments, the CSTDPPC permits a high concentration of payload molecules to be delivered to the internal and/or external spaces of targeted cells. In some embodiments, specific groupings, organizations, or cell types are targeted.

[0056] A payload-to-cell-surface-targeting domain (“PCSTD”) ratio as used herein refers to the average number of payload molecules conjugated to a single cell-surface- targeting-domain-polymer-payload conjugate (CSTDPPC).

-l i [0057] A “drug-to-antibody ratio” (DAR) as used herein is the average number of drug molecules conjugated to a single monoclonal antibody, e.g., as measured by sizeexclusion chromatography.

[0058] “Payload” as used herein refers to a molecule intended to be targeted and/or delivered to the intracellular space and/or the extracellular space around targeted cells. The purposes and/or functions of payloads, without limitations, include therapy, cytotoxicity, cell identification, cell localization, cell sorting, and/or cell isolation.

[0059] “Oligonucleotide” as used here in refers, without limitation, to polynucleotide chains comprising DNA, RNA and their analogs that can be single or double stranded. Examples of oligonucleotides include, but are not limited to siRNAs, shRNAs, microRNAs, ASOs, gapmers, mixmers, PMOs, aptamers, antagomirs, agomirs. In some embodiments, an oligonucleotide may contain one or more chemical modifications including but not limited to modifications of ribose (LNA, UNA, GNA, CeNA, HNA, 2’0-Me, 2’0- MOE, 2’F and 4’ thioribonucleosides), modifications of phosphate (Tert-butyl-S-acyl-2- thioethyl, boranophosphate, 5’(E)-vinylphosphonate, phosphorothioate), and modifications of base (pseudouridine, 2’thiouridine).

[0060] A “cell-surface-targeting-domain (CSTD)” as used herein refers without limitation to molecules that bind to or have affinity for molecules on the surface of cells and can be comprised without limitations to all forms and modified variations of proteins, peptides, aptamers, amino acids, lipids, nucleic acids, small molecules and combinations thereof. As used herein, CSTDs have sufficient affinity for their targets to permit efficacious amounts of payload components to reach cells targeted by cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) and to be subsequently internalized. In some embodiments, the CSTDPPC is internalized upon binding of the CSTD to the cell surface target (CST). In some embodiments, the payload is released from the CSTTDPPC when the CSTD binds or is bound to the CST and the payload or components linked to the payload then drive internalization. A CSTD comprising a protein will still comprise a CSTD if one or more amino acid is added to or deleted from the CSTD. A CSTD comprising nucleotides, lipids, carbohydrate polymers, or a small molecule is still a CSTD if additions or deletions are made to the structure or composition. For example, a CSTD comprising nucleotides is still a CSTD if one or more nucleotides is added or deleted or if a modification is added to or deleted from the nucleotide. [0061] A “cell surface target (CST)” as used herein refers, without limitation, to molecules comprising or associated with the cell membrane that are accessible to the cell- surface-targeting domain (CSTD) of the cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) from the extracellular space. CSTs can be comprised of, without limitation, proteins, protein modifications, receptors, lipids, modified lipids, phospholipids, sugar moieties, aptamers, nucleotides, and cell free DNA and RNA. In some embodiments, a cell surface target comprises an engineered molecule that binds to or interacts with a cell surface target. In some embodiments, the CSTD of the CSTDPPC targets more than one CST.

[0062] An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion or fragment thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen binding portions include, for example, Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG , IgG?, IgG4, IgAi and IgA?. The heavy -chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0063] As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein, “humanized” antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0064] “Anti-VEGFR2-antibody” as used herein refers to IgG molecules that bind the vascular endothelial growth factor receptor 2 (VEGFR2).

[0065] “Tissue specific” as used herein refers to groupings of cells that exhibit similar characteristics or an organization that permits the grouping of cells to produce a specific function or functions. Tissues as used herein refer without limitation to organizations of cells in the eye, heart, brain, lung, kidney, liver, muscle, nervous system, skin, pancreas, bone, bladder, stomach, intestine, colon, uterus, ovary, testes, and thyroid.

[0066] A “surface of a cell” as used herein refers to regions of a cell exposed to the extracellular space or in contact with other cells. In some embodiments, surface of a cell comprises the exterior lipid bilayer of the cell membrane. In some embodiments, the surface of the cell comprises proteins, including transmembrane protein, and sugar moieties.

[0067] “Internalization” as used herein refers without limitation to the transfer of a molecule or molecules from the extracellular space to the internal spaces of cells, which is encapsulated by the cell membrane. In some embodiments, a payload is internalized. Internal spaces in cells refer without limitation to structures and compartments including the nucleus, cytoplasmic organelles and mitochondria. Internalization can occur through active or passive mechanisms including but not limited to endocytosis and diffusion. In some embodiments, internalization is initiated by interactions between components of the CSTDPPC and a target or targets on the cell surface. In some embodiments, interactions between an antibody and a receptor on the cell surface induce a receptor-mediated endocytosis response that will result in the internalization of antibody and conjugate payload. In some embodiments, a payload induces its own internalization.

[0068] “Payload-to-cell-surface-targeting domain ratio” or “PCSTD ratio” as used herein refers to the average number of payload molecules conjugated to the CSTDPPC. In some embodiments the PCSTDR is regulated or controlled by the number of conjugatable sites in the polymer.

[0069] The term "subject" as used herein includes human and mammalian subjects that receive either prophylactic or therapeutic treatment.

[0070] “Polymer” refers to a series of monomer groups linked together. A polymer is composed of multiple units of a single monomer (a homopolymer) or two or more different monomers (a heteropolymer or a copolymer). Polymers are prepared from monomers that include, but are not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinylpyridine, vinylpyrrolidone and vinyl esters such as vinyl acetate. Additional monomers are useful in high MW polymers. When two or more different monomers are used to form a single polymer, such polymer is a “copolymer.” The two or more monomers are “comonomers” that copolymerized to form a copolymer. The polymer can be linear or branched. When the polymer is branched, each polymer chain is referred to as a “polymer arm.” The end of the polymer arm linked to the initiator moiety is the proximal end, and the growing-chain end of the polymer arm is the distal end. On the growing chain-end of the polymer arm, the polymer arm end group can be the radical scavenger, or another group. The copolymer may be a block copolymer, an alternating copolymer, a gradient copolymer, or a random copolymer (i.e., statistical copolymer).

[0071] “Block copolymer” refers to a copolymer consisting of two or more distinct blocks of homopolymer.

[0072] “Alternating copolymer” refers to a copolymer comprising two or more monomer units distributed in alternating sequence.

[0073] Gradient copolymer” refers to a type of copolymer in which composition of the monomer units varies gradually along the polymer chain.

[0074] Star copolymer” refers to a type of branched polymer with a general structure consisting of several linear chains connected to a central core. Each linear chain is a polymer arm.

[0075] ‘Random copolymer” refers to a polymer having at least two different monomer groups that are distributed randomly (with uncertain sequences of comonomer units) throughout the polymer backbone, such as a polymer arm. The random copolymer may or may not be completely random in a statical sense and may include one or more blocks of repeating units of a single monomer species, one or more sections containing alternating units of different monomer species, and/or comonomers linked together in any sequence. The random copolymers are prepared from monomers that include, but are not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridine and vinyl-pyrrolidone. Additional monomers are useful in forming a random copolymer.

[0076] “Zwitterionic moiety” refers to a compound having both a positive and a negative charge. Zwitterionic moieties useful in the random copolymers can include a quaternary nitrogen and a negatively charged phosphate, such as phosphorylcholine (PC): wherein * denotes the point of attachment, for example, to a monomer. The phosphorylcholine is a zwitterionic group and includes salts (such as inner salts), and protonated and deprotonated forms thereof. In some embodiments, other zwitterionic moieties are useful in the random copolymers.

[0077] “Phosphoryl choline containing polymer” is a polymer that contains a phosphorylcholine, such as a polymer containing at least one monomer unit that includes a phosphorylcholine. “Zwitterion containing polymer” refers to a polymer that contains a zwitterion, such as a polymer containing at least one monomer unit that includes a zwitterion.

[0078] An “initiator” is a compound capable of serving as a substrate on which one or more polymerizations can take place using monomers or comonomers as described herein. The polymerization can be a conventional free radical polymerization or in some embodiments a controlled/”living” radical polymerization, such as Atom Transfer Radical Polymerization (ATRP), Reversible Addition Fragmentation Termination (RAFT) polymerization or nitroxide mediated polymerization (NMP). The polymerization can be a “pseudo” controlled polymerization, such as degenerative transfer. Initiators suitable for ATRP contain one or more labile bonds which can be homolytically cleaved to form an initiator fragment, I, being a radical capable of initiating a radical polymerization, and a radical scavenger, I’, which reacts with the radical of the growing polymer chain to reversibly terminate the polymerization. In some embodiments, the radical scavenger I’ comprises a halogen, but can also be an organic moiety, such as a nitrile. Also provided herein, the initiator can contain one or more 2- bromoisobutyrate groups as sites for polymerization via ATRP.

[0079] “Linker” refers to a chemical moiety that links two groups together. The linker can be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH sensitive, photolabile, or disulfide linkers, among others. Other linkers include homobifunctional and heterobifunctional linkers. Linker as used herein can also refer to a moiety connecting a payload or a cell surface targeting domain (CSTD) to a polymer. In some embodiments, a linker is a stable, or degradable covalent bond. Nonlimiting examples of the linker formation from reactions between a linking group on the polymer and the reactive group on the payload or CSTD include those illustrated in Table 1.

Table 1: Some non-limiting examples of the reaction of the linking groups and some groups typically found or introduced into functional agents.

[0080] “Hydrolyzable linker” refers to a chemical linkage or bond, such as a covalent bond, that undergoes hydrolysis under physiological conditions. The tendency of a bond to hydrolyze may depend not only on the general type of linkage connecting two central atoms between which the bond is severed, but also on the substituents attached to these central atoms. Non-limiting examples of hydrolytically susceptible linkages include esters of carboxylic acids, phosphate esters, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, and some amide linkages.

[0081] “Enzymatically cleavable linker” refers to a linkage that is subject to degradation by one or more enzymes. Some hydrolytically susceptible linkages may also be enzymatically degradable. For example esterases may act on esters of carboxylic acid or phosphate esters, and proteases may act on peptide bonds and some amide linkages.

[0082] “pH sensitive linker” refers to a linkage that is stable at one pH and subject to degradation at another pH. For example, the pH sensitive linker can be stable at neutral or basic conditions, but labile at mildly acidic conditions.

[0083] “Photolabile linker” refers to a linkage, such as a covalent bond, that cleaves upon exposure to light. The photolabile linker includes an aromatic moiety in order to absorb the incoming light, which then triggers a rearrangement of the bonds in order to cleave the two groups linked by the photolabile linker.

[0084] “Functional agent” is defined to include a bioactive agent or a diagnostic agent. A "bioactive agent" is defined to include any agent, drug, compound, or mixture thereof that targets a specific biological location (targeting agent) and/or provides some local or systemic physiological or pharmacologic effect that can be demonstrated in vivo or in vitro.

[0085] Non-limiting examples include drugs, vaccines, antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes, etc).

[0086] A "diagnostic agent" is defined to include any agent that enables the detection or imaging of a tissue or disease. Examples of diagnostic agents include, but are not limited to, radiolabels, fluorophores and dyes.

[0087] “Therapeutic protein” refers to peptides or proteins that include an amino acid sequence which in whole or in part makes up a drug and can be used in human or animal pharmaceutical applications. Numerous therapeutic proteins are known to practitioners of skill in the art including, without limitation, those disclosed herein.

[0088] “Poly(acryloyloxyethyl phosphoryl choline) containing polymer” refers to a polymer of acrylic acid containing at least one acryloyloxy ethyl phosphorylcholine monomer such as 2-methacryloyloxy ethyl phosphorylcholine (i.e., 2-methacryloyl-2 '- trimethyl ammonium ethyl phosphate).

[0089] “Contacting” refers to the process of bringing into contact at least two distinct species such that they can react. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0090] “Water-soluble polymer” refers to a polymer that is soluble in water. In some embodiments, a solution of a water-soluble polymer may transmit at least about 75%, more or at least about 95% of light, transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer or segment thereof may be at least about 35%, at least about 50%, about 70%, about 85%, about 95% or 100% (by weight of dry polymer) soluble in water.

[0091] ‘Molecular weight” (MW) in the context of the polymer can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight average molecular weight. The polymeric reagents of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low poly dispersity values of in some embodiments, less than about 2, as judged by gel permeation chromatography.

[0092] In some embodiments the polydispersities may be in the range of about 1.5 to about 1, about 1.4 to about 1, about 1.3 to about 1, about 1.2 to about 1, about 1.5 to about 1.2, about 1.4 to about 1.2. In some embodiments the polydispersities may be less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, less than about 1.15, in some embodiments less than about 1.1, in some embodiments less than about 1.05, and in some embodiments less than about 1.03, or less than 1, in some embodiments less than about 2.

[0093] ‘Protected,”, “protected form”, “protecting group” and “protective group” refer to the presence of a group (i.e., the protecting group) that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule, if any. The skilled artisan will recognize protecting groups known in the art, such as those found in the treatise by Greene et al., "Protective Groups In Organic Synthesis," 3rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0094] “Spacer,” and “spacer group” are used interchangeably herein to refer to an atom or a collection of atoms used to link interconnecting moieties such as a terminus of a water-soluble polymer and a reactive group of a functional agent and a reactive group. A spacer may be hydrolytically stable or may include a hydrolytically susceptible or enzymatically degradable linkage.

[0095] “Alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together.

[0096] The term "lower" referred to above and hereinafter in connection with organic radicals or compounds respectively defines a compound or radical which can be branched or unbranched with up to and including 7, preferably up to and including 4 and (as unbranched) one or two carbon atoms.

[0097] “Alkylene” refers to an alkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene. For instance, a straight chain alkylene can be the bivalent radical of -(CH2)n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.

[0098] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: -OR', =0, =NR', =N-0R', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', - CO₂R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)₂R', -NH- C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)₂R', -S(O)₂NR'R", -CN and -NO₂ in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R', R" and R'" each independently refer to hydrogen, unsubstituted (Ci- Cs)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Ci-C4)alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7- membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., - C(0)CH3, -C(O)CF3, -C(O)CH₂OCH3, and the like). Preferably, the substituted alkyl and heteroalkyl groups have from 1 to 4 substituents, more preferably 1, 2 or 3 substituents. Exceptions are those perhalo alkyl groups (e.g., pentafluoroethyl and the like) which are also preferred and contemplated by the present invention.

[0099] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR', =0, =NR', =N-0R', -NR'R", -SR', -halogen, - SiR’R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(0)R', -NR'- C(0)NR"R"', -NR"C(0)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', - S(O)₂R', -S(0)₂NR'R", -NRS0₂R', -CN and -NO₂ in a number ranging from zero to (2m'+ 1), where m' is the total number of carbon atoms in such radical. R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(0)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0100] “Alkoxy” refers to alkyl group having an oxygen atom that either connects the alkoxy group to the point of attachment or is linked to two carbons of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2- butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. For example, the alkoxy groups can be substituted with halogens to form a "halo-alkoxy" group.

[0101] “Carboxyalkyl” means an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" means an cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl means an alkyl group (as defined herein) substituted with an alkoxy group. The term "carboxy" employed herein refers to carboxylic acids and their esters.

[0102] “Haloalkyl” refers to alkyl as defined above where some or all of the hydrogen atoms are substituted with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or radical which has all available hydrogens that are replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1 -trifluoromethyl, and perfluoromethoxy refers to 1,1,1 -trifluoromethoxy.

[0103] “Fluoro-substituted alkyl” refers to an alkyl group where one, some, or all hydrogen atoms have been replaced by fluorine.

[0104] The phrase "a" or "an" entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0105] ‘About” as used herein means variation one might see in measurements taken among different instruments, samples, and sample preparations. EMBODIMENTS

[0106] Provided herein are cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) that comprises a cell-surface-targeting domain (CSTD), a polymer, and one or more payload. In some embodiments, the CSTD is linked to the polymer and the polymer is also linked to one or more payloads(s). In some embodiments, the one or more payload(s) may be linked to the CSTD. In some embodiments, the one or more payload(s) may be linked to the CSTD and the polymer (e.g., some payloads are linked to the CSTD, and other payloads are linked to the polymer). These molecules are capable of targeting cells and delivering payloads to their internal spaces (e.g., intracellular spaces). When the cell-surface- targeting domain (CSTD) is bound to the cell surface target (CST) on the surface of the cell, it is internalized into the cell. In some embodiments, the CSTDPPCs are capable of delivering payloads to the external spaces of targeted cells.

[0107] In some embodiments, the polymer may be a linear polymer or a multi-arm polymer (e.g., any number of arms from 1 to 100). In some embodiments, the multi-arm polymer may be a branched polymer. In some embodiments, the polymer may be a copolymer formed by two or more monomers. In some embodiments, each of the polymer arm(s) may be a copolymer arm. In some embodiments, the copolymer may be a random copolymer. In some embodiments, the polymer may be a homopolymer.

[0108] Provided herein also include methods of delivering payloads to the internal spaces of cells or the internal spaces of cells within subjects. In some embodiments, the cell- surface-targeting-domain-polymer-payload conjugate CSTDPPC is administered to a cell. In some embodiments, the CSTDPPCs is administered to a cell within a subject. In some embodiments, the CSTDPPC is delivered to the targeted tissue by injection. In some embodiments, the CSTDPPC is delivered to the targeted cell type via diffusion and/or circulation through the subject. In some embodiments, the CSTDPPC is delivered to the targeted cell by first binding to a circulating cell within the subject wherein the circulating cell then delivers the CSTDPPC to the target cell. In some embodiments, the targeted delivery of the CSTDPPC to specific cells, cell surface targets (CTSs), and/or subcellular compartments is a key step in a payload eliciting a biological response.

[0109] In some embodiments, the cell-surface-targeting domain CSTD binds to the CST and when the CSTD is bound to the CST on the surface of the cell, it is internalized into the cell. In some embodiments, binding of the CSTD to the CST initiates internalization of CSTDPC. In some embodiments, the payload is released from the CSTDPPC in the internal spaces of cells. In some embodiments, the CSTDPPC comprises a linker (e.g., a cleavable linker) between the payload and the second monomer configured to release the payload in the cell upon internalization following binding of the CSTD to the one or more targets on the surface of the cell.

[0110] An embodiment of cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) 100 that allow payloads 400 to be internalized into cells is depicted in FIG. 1. The CSTDPPC 100 comprises a cell-surface-targeting domain (CSTD) 200 that is linked to the polymer 300 and the polymer 300 is linked to the payloads 400. When the CSTD 200 is bound to the CST 500 on the surface of the cell, the payload 400 is released from the polymer 300 and the payload 400 drives internalization into the intracellular space 800 of the cell thereby internalizing the payload 400. In some embodiments, the payload 400 comprises a peptide that drives internalization. In some embodiments, the payload 400 comprises a molecule linked to a peptide that drives internalization thereby internalizing the payload molecule. In some embodiments, the payload 400 is released from the peptide inside the cell after internalization. In some embodiments, the payload remains linked to the peptide after internalization. In some embodiments, the payload is linked to the polymer.

[oni] In some embodiments, with reference to FIG. 1, methods are provided for delivering cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) comprising payloads capable of driving internalization. In some embodiments, a CSTDPPC is administered to cells or cells within a subject. In some embodiments, the CSTD 200 binds to one or more CST 500. In some embodiments, CSTD 200 binding to the CST 500 results in the payload 400 being released from the polymer wherein when the payload 400 is released the payload 400 drives internalization into the cell 800. In some embodiments, the payload is conjugated to a molecule that drives internalization of itself and the payload. In some embodiments, the molecule that drives internalization is a peptide.

[0112] FIG. 6 depicts another embodiment of CSTDPPC where the payload is linked to the CSTD. In this embodiment, the CSTD is an antibody 201 that links to the polymer 300, the payload is a label/dye (e.g., fluorescent dye) 401 linked to the antibody 201. When the antibody 201 binds to the CST 501, the payload 401 is internalized into the intracellular space 800. Such CSTDPPC is an example of the cell-surface-targeting-domain-polymer- fluorescent-dye conjugate (CSTDPFDC) 101 for delivering a fluorescent dye into internal spaces of cells . In some embodiments, internalization of a fluorescent dye 401 is achieved by providing a CSTDPFDC 101 comprising a CSTD that comprises an anti-VEGFR2 201, a polymer 300, and a fluorescent dye 401 conjugated to the antibody 201. In some embodiments, the CSTDPFDC is internalized and permits the cell to be visualized and identified. In some embodiments the internalized CSTDPFDC permits specific cellular compartments and /or organelles to be visualized.

[0113] In some embodiments, with reference to FIG. 6, a cell-surface-targetingdomain-polymer-fluorescent dye conjugate (CSTDPFDC) 101 comprises a cell-surface- targeting domain (CSTD) comprising an antibody (e.g., an anti-VEGFR2 antibody) 201, a polymer 300 conjugated to the antibody, and a fluorescent dye 401 conjugated to the antibody 201 and/or the polymer. In some embodiments, the polymer 300 comprises a homopolymer. In some embodiments, the polymer 300 comprises a copolymer. In some embodiments, the polymer 300 is conjugated to the antibody 201. In some embodiments, the fluorescent dye 401 is covalently linked to the antibody 201. In some embodiments, the fluorescent dye 401 is covalently linked to the polymer. The CSTD can be any suitable CSTD as described herein. The antibody can be any suitable antibody as described herein. In some embodiments, polymer includes a monomer that includes a zwitterionic moiety. In some embodiments, polymer includes a monomer selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone. In some embodiments, the polymer is any one of the copolymers described herein.

[0114] In some embodiments, cell-surface-targeting domains (CSTDs) have sufficient affinity for their CSTs to permit binding and subsequent internalization.

[0115] In some embodiments, with reference to FIG. 1, cell-surface-targeting domains (CSTDs) 200 bind to one or more cell-surface target (CST) 500 on the surface of a cell. In some embodiments, cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPCs) 100 comprise one or more cell-surface-targeting domains (CSTDs) 200 that can bind to one or more cell surface targets (CSTs) 500. In some embodiments, the CSTD 200 comprises proteins, oligonucleotides, aptamers, macrocycles, constrained peptides, sugar moi eties, lipids or combinations thereof. In some embodiments, the CSTD comprises a protein selected from a group consisting of antibodies, single-chain Fvs, Fabs, single domain antibodies, centyrins, nanofitins, affibodies, DARPins, affilins, anticalins, Kunitz, avimers, monobodies, peptides, cyclic peptides, trap domains, trap-domain-antibody fusions, knottins, and native and engineered proteins that bind a target or targets on the surface of a cell or combinations thereof. In some embodiments, the CSTD comprises an aptamer. In some embodiments the CSTD 200 comprises a full-length native protein. In some embodiments, the CSTD 200 comprises part of a full-length native protein. In some embodiments, the CSTD 200 comprises an antibody. In some embodiments, the CSTD comprises an anti-VEGFR2- antibody. In some embodiments, the CSTD comprises one or more TRAP domains. In some embodiments, the CSTD comprises an antibody-TRAP fusion protein. In some embodiments, the CSTD inhibits a biological process. In some embodiments, a protein CSTD still comprises a CSTD if one or more amino acids are added or deleted from it.

[0116] In some embodiments, the Cell Surface Target (CST) comprises, proteins, sugar moieties, and or lipids. In some embodiments, protein CSTs comprise receptors, channels, adhesion molecules, antigen presented on immune cells, transporters, transmembrane proteins. In some embodiments, the CSTs comprise proteins that are modified and or oxidized. In some embodiments the cell-surface-targeting domain (CSTD) binds a CST that comprises a modification or modifications. Suitable CSTs include, without limitation, VEGFR2, VEGFR1, EGFR, B7-H3, FRa, HER2, HER3, TROP2, EphA2, Claudin 18.2, c- MET, ASGPR, TF, Nectin-4, CD22, SIRPoc, CD30, CD79b, SSEA4, CD33, CD74, CD56 (NCAM), CanAg, CA6, CD 138, mesothelin, a(v) integrin, CD 19, gpNMB, CD70, CD37, EGFRvIII, TfRl, CD4, ACE2, PTK7, siglec-5, IGHM, L1CAM, Annexin A2, CD71. In some embodiments, CSTDs bind protein modifications. In some embodiments, CSTDs bind lipid modifications. In some embodiments, CSTDs include, without limitation, Anti-VEGFR2 , Anti-VEGFRl, Anti-EGFR, Anti-B7-H3, Anti-Folate receptor alpha (FRa), Anti-HER2, Anti- HER3, Anti-TROP2, Anti-EphA2, Anti-Claudin 18.2, Anti-c-MET, Anti-ASGPR, AntiTissue Factor (TF), Anti-Nectin-4, Anti-CD22, Anti-SIRPa, Anti-CD30, Anti-CD79b, Anti- SSEA4, Anti-CD33, Anti-CD74, Anti-CD56, Anti-CanAg, anti-CA6, anti-CD138, anti- mesothelin, anti-a(v) integrin, anti-CD19, anti-gpNMB, anti-CD70, anti-CD37, anti- EGFRvIII, Anti-TfRl, anti-PTK7, anti-siglec-5, anti-IGHM, anti-LlCAM, anti-Annexin A2, anti-CD71, HIV env, COVID-19 spike protein, N-acetylgalactosamine. [0117] In some embodiments, the Cell-Surface-Targeting Domain-Cell Surface Target (CSTD-CST) interaction comprises an interaction between a receptor and a receptor ligand. In some embodiments, the CSTD-CST interaction comprises an interaction between a receptor and a viral protein. In some embodiments, the CSTD-CST interaction comprises an interaction between a receptor and a nucleic acid, sugar, and/or lipid.

[0118] In some embodiments, the CSTD is or includes an antibody. Any suitable antibody can be used as the CSTD. In some embodiments, the antibody binds to a CST and is internalized upon binding. In some embodiments, the CSTD is or includes an antibody such as, without limitation, MC813-70 or chMC813-70 (Anti-SSEA4), OBI-998 (Anti-SSEA4), gemtuzumab (anti-CD33), inotuzumab (anti-CD22), milatuzumab (anti-CD74), lorvotuzumab (anti-CD56), cantuzumab (anti-CanAg), huDS6 (anti-CA6), indatuximab (anti-CD138), anetumab (anti-mesothelin), intetumumab (anti-a(v) integrin), coltuximab (anti-CD19), brentuximab (anti-CD30), glembatumumab (anti-gpNMB), vorsetuzumab (anti-CD70), enfortumab (anti-nectin-4), trastuzumab (anti-HER2), naratuximab (anti-CD3 ), depatuxizumab (anti-EGFR), AMG-595 (anti-EGFRvIII), ramucirumab (anti-VEGFR2), DC101 (anti-VEGFR2), icrucumab (anti-VEGFRl), trastuzumab (anti-HER2), disitamab (anti-HER2), HLX22 (anti-HER2), pertuzumab (anti-HER2), INA01 (anti-TfRl). In some embodiments, the CSTD is or includes an aptamer. Any suitable aptamer can be used as the CSTD. In some embodiments, the aptamer binds to a CST and is internalized upon binding. In some embodiments, the CSTD is or includes an aptamer such as, without limitation, sgc8 (anti-PTK7), KI 9 (anti-siglec-5), TD05 (anti-IGHM), ylyl2 (anti-LlCAM), ACE4 (antiAnnexin A2), XQ-2d (anti-CD71), H02 (anti-integrin avpi). In some embodiments, the CSTD is or includes an anti-TfRl aptamer selected from: tJBA8.1, HG1-9, GS24, FB4, DW4, C2.min, and Waz. In some embodiments, the CSTD is or includes macrocycle (e.g., macrocyclic peptide). Any suitable macrocycle (e.g., macrocyclic peptide) can be used as the CSTD. In some embodiments, the macrocycle (e.g., macrocyclic peptide) binds to a CST and is internalized upon binding. In some embodiments, the CSTD is or includes a macrocycle (e.g., macrocyclic peptide) such as, without limitation, Pasireotide (anti-somatostatin).

[0119] The CSTD can be linked or conjugated to the polymer or copolymer in any suitable manner. In some embodiments, the CSTD (e.g., an antibody) includes a cysteine with an accessible thiol side chain that is conjugated to the copolymer via maleimide-cysteine chemistry. Tn some embodiments, the CSTD (e.g., an aptamer) includes a 5’ thiol group that is conjugated to the copolymer via maleimide-thiol chemistry.

[0120] In some embodiments, the cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprises one or more payloads that has biological activity. In some embodiments, the payload comprises molecules that are precursors to molecules with biological activity. In some embodiments, the payload precursors are converted into an active molecule by endogenous or engineered enzymes after the CSTDPPC contacts the targeted cell and/or is internalized. In some embodiments, the payload has chemical groups that block biological activity and that are cleaved or modified by endogenous or engineered enzymes after the CSTDPPC contacts the targeted cell and/or is internalized. In some embodiment, the payload does not have biological activity.

[0121] Any suitable payload can be used in the CSTDPPC. In some embodiments, the payload comprises a molecule selected from the group comprising oligonucleotides, small molecules, proteins, peptides, aptamers, macrocycles, constrained peptides, cyclic peptides, tRNA or combinations thereof. In some embodiments, the payload is or includes a protein synthesis inhibitor (e.g., puromycin, etc.), transcription inhibitor (e.g., alpha amanitin, actinomycin D, etc.), translation inhibitor (e.g., puromycin, etc.), DNA damaging agent (e.g. calicheamicin, PBD dimer, etc.), microtubule inhibitor (e.g., auristatins, maytansines, etc.), topoisomerase inhibitors (e.g. exatecan, deruxtecan, etc ), immunomodulator, antimetabolite (e.g. methotrexate, etc ), antimitotic (e.g., paclitaxel, irinotecan, etc.), proteolysis-targeting chimeras (PROTACs), polyamine inhibitors, and/or a polyamine transport inhibitor, or a functional derivative(s) thereof. In some embodiments, the payload is or includes a tubulin inhibitor, such as but not limited to, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some embodiments, the payload is or includes an oligonucleotide. In some embodiments, the payload is or includes an oligonucleotide, such as but not limited to, siRNA, shRNA, miRNA, or antisense oligonucleotide (ASO), gapmer, mixmer, PMO, antagomir, or agomir configured to target a gene of interest. Any suitable gene of interest can be targeted by the oligonucleotide payload. In some embodiments, the gene of interest is selected from and not limited to: VEGF, Connexin-43, IRS-1, NRARP, CEP290, USH2A, VEGFR1, VEGFR2, CTGF, TGF-beta2, RTP801, miR-33, DMPK, DUX4, Exon-44, Exon- 51, PCSK9, cMyc, p53, STAT3, KRAS (including KRAS-G12D), TLR9, TLR7, TLR8, EphA2, HIF-2a, HIF-loc, GSTP, PKN3, PLK-1, RRM2, PD1, PD-L1.

[0122] In some embodiments, the CSTDPPC includes one payload (e.g., a single type of payload). In some embodiments, the payload comprises combinations of two or more different types of payload molecules. In some embodiments, the two or more different types of payload molecules include a combination of peptides and oligonucleotides. In some embodiments, the payload comprises combinations of two or more of the same type of payload molecule with a non-limiting example comprising two or more different small molecules with different or the same biological activity or activities and/or target or targets. In some embodiments, the payload comprises an oligonucleotide. In some embodiments, the payload comprises combinations of oligonucleotides with two or more different sequence identities. In some embodiments, the oligonucleotide (e.g., siRNA or ASO) is modified to increase stability and/or targeting. Any suitable number of payloads can be included in the CSTDPPC. In some embodiments, the CSTDPPC includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of payloads. In some embodiments, the CSTDPPC includes 2 payloads (e.g., 2 different types of payloads). In some embodiments, the CSTDPPC includes 3 payloads (e.g., 3 different types of payloads). In some embodiments, the CSTDPPC includes 4 payloads (e.g., 4 different types of payloads).

[0123] In some embodiments, the payload is capable of being detected. In some embodiments, the payload is a fluorescent dye.

[0124] In some embodiments, with reference to FIG. 1, the payload 400 is released from the cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) 100 into the internal cellular spaces when the CSTDPPC 100 is internalized. In some embodiments, the released payload 400 is capable of diffusing, being transported and/or being encapsulated by proteins and/or vesicles within the cell independently of the polymer and/or the cell-surface- targeting domain (CSTD) 200. In some embodiments, the payload 400 is capable of exerting its biological function or activity independent of the polymer 300 and/or the CSTD 200. In some embodiments, the payload 400 is released from the CSTDPPC through proteolytic cleavage that occurs in the external and/or internal spaces of cells. In some embodiments, the payload 400 is linked to the polymer 300 through a pH-sensitive linker that is cleaved when the CSTDPPC 100 is internalized and/or encounters a change in pH. In some embodiments, the pH change that cleaves the linker occurs upon binding to the targeted cells or localization to targeted tissue. In some embodiments, the payload 400 is linked to the polymer 300 through a reducible linker that is cleaved when the CSTDPPC 100 is internalized and/or a change in the reduction potential. In some embodiments, the reduction potential change that cleaves the linker occurs upon binding to the targeted cells or localization to targeted tissue.

[0125] In some embodiments, with reference to FIG. 1, the payload 400 is modified and/or processed in the internal cellular spaces and becomes biologically active. In some embodiments, the payload 400 modified and/or processed in the internal cellular spaces is secreted into the extracellular space where is exerts a biological function. In some embodiments, the payload 400 is not released from the cell-surface-targeting-domain-polymer- payload conjugate (CSTDPPC) 100 when the CSTDPPC 100 is internalized.

[0126] In some embodiments, the payload has a therapeutic effect on the targeted cells and/or cells of a subject. In some embodiments, the therapeutic response comprises degrading coding mRNAs and/or noncoding RNAs expressed from specific endogenous genes and/or genomic regions. In some embodiments, mRNA expressed from viral genes and/or regions of viral genomes or integration sites are targeted. In some embodiments, the payload targets expressed viral genes integrated into an endogenous genome or maintained in cells in a latent state for degradation. In some embodiments, the payload activates latent viral genes. In some embodiments, the payload blocks the translation of mRNAs into proteins. In some embodiments, the payload blocks RNA modifications, degradation and/or formation of secondary and tertiary structures. In some embodiments, the payload targets mRNA expressed from a gene or genomic region that comprises germ line and/or somatic cell mutations for degradation. In some embodiments, the payload blocks the active site of enzymes. In some embodiments, the payload blocks interactions between targets and ligands. In some embodiments, payloads stabilize interactions between targets and ligands. In some embodiments, the payload interaction with target results in target degradation. In some embodiments, the payload prevents proteins from folding or being transported to their sites of function. In some embodiments, the payload comprises a combination therapy comprising drugs that work by different mechanisms to produce a therapeutic and/or synergistic effect or to prevent the development of drug resistance. In some embodiments, the payload initiates a response from the immune system. In some embodiments, the immune response confers immunity to an infectious agent to the subject or prevents the develop of a malignancy caused by an infectious agent or a somatic cell mutation. In some embodiments, the payload permits targeted cells to be identified, sorted and/or isolated.

[0127] In some embodiments, the cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprises a cell-surface targeting domain (CSTD) that targets the CSTDPPC to the surface of one or more cells. In some embodiments, the targeted cells are grown in vitro. In some embodiments, the targeted cells are used for research purposes. In some embodiments, the targeted cells are grown for therapeutic purposes and comprise allografts and/or autografts. In some embodiments the cell-surface-targeting domain (CSTD) targets the CSTDPPC to cells comprising a specific tissue. In some embodiments, targeted tissues include but are not limited to the eye, heart, kidney, pancreas, skin, lung, and brain. In some embodiments, the CSTD targets a single cell or a subset of cells within a specific tissue. In some embodiments, the CSTD targets different cell types residing in different tissues but having the same cell surface target. In some embodiments, the targeted cells interact with an extracellular matrix. In some embodiments, the targeted cells are circulating cells.

[0128] In some embodiments, with reference to FIG. 1, cell-surface-targeting domain (CSTD) 200 binding to a cell surface target (CST) 500 initiates internalization of the CSTDPPC. In some embodiments, the cell-surface-targeting domain (CSTDPPC) 100 is internalized through endocytosis including but not limited to clathrin-mediated endocytosis, receptor-mediated endocytosis, caveolae-mediated endocytosis, caveolae lipid-raft-mediated endocytosis, micropinocytosis, and macropinocytosis. In some embodiments, the CSTDPPC 100 undergoes transcytosis. In some embodiments, the CSTDPPC 100 is internalized by directly penetrating the cell membrane. In some embodiments, the CSTD 200 comprises a viral protein that binds to a specific cell surface target and the CSTDPPC 100 is internalized through the mechanism used by viruses to be internalized. In some embodiments, internalization of the CSTDPPC 100 lyses the cell. Any or all of these purposes or functions can be applied to cells grown in vitro, cells in an organism, cells in a subject or cells used for laboratory research.

[0129] In some embodiments, with reference to FIG. 1, the payload 400 is released into the extracellular space when the cell-surface-targeting domain (CSTD) 200 binds to or when the CSTD 200 is bound to its cell surface target (CST) 500. In some embodiments, the released payload 400 is internalized via a channel. In some embodiments, the payload 400 is linked to a cell penetrating peptide that internalizes the payload when it is released from the polymer. In some embodiments, the payload 400 is not released into the extracellular space when the CSTD 200 binds to or is bound to its target.

[0130] In some embodiments, the polymer in a CSTDPPC comprises a copolymer (e.g., a random copolymer). In some embodiments, the copolymer (e.g., random copolymer) comprises multiple polymer arms extending from a branched initiator fragment. In some embodiments, the copolymer (e.g., random copolymer) is a linear polymer comprising one arm. In some embodiments, each polymer arm comprises a first monomer and a second monomer distributed randomly throughout the copolymer. In some embodiments, the first monomer further comprises a zwitterionic moiety, and the second monomer is linked to the payload 400. In some embodiments, the polymer in the CSTDPPC comprises a block copolymer.

[0131] In some embodiments, each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone.

[0132] In some embodiments, the copolymer (e.g., random copolymer) is represented by Formula I:

In formula I, the monomer units M¹ and M² are any monomers suitable for polymerization via controlled free radical methods, such as atom-transfer radical polymerization (ATRP). In some embodiments, each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone. In some embodiments, M² links to the payload. In some embodiments, M² links to CSTD. ZW is a zwitterionic moiety. I is an initiator fragment. In some embodiments, initiator fragment I conjugates with the cell-surface-targeting domain. I’ is a radical scavenger, such that the combination of I-F is an initiator, I¹, for the polymerization of the random copolymer of Formula I. Subscripts x and y¹ are each independently an integer of from 1 to 1000. Subscript z is an integer of from 1 to 10. Subscript s is an integer of from 1 to 100. Subscript n is an integer of from 1 to 20. [0133] In some embodiments, the random copolymer is further represented by Formula II: (Formula II); wherein each of R¹ and R² is H or 1-4 alkyl, and PC is phosphorylcholine. I is an initiator fragment that conjugates with the cell -surface-targeting domain 200. I’ is a radical scavenger as defined herein. Subscripts x and y¹ are each independently an integer of from 1 to 1000. Subscript s is an integer of from 1 to 100. Subscript n is an integer of from 1 to 20.

[0134] In some embodiment, with reference to FIG. 1, the second monomer is linked to the payload 400. In some embodiments, the payload 400 is directly linked to the second monomer. In some embodiments, the payload 400 is indirectly linked to the second monomer. In some embodiments, the linker that links the payload to the second monomer is stable. In some embodiments the linker that links the payload to the second monomer is cleavable. In some embodiments, the linker is configured (e.g., includes a protease-labile, pH- sensitive, or reducible cleavage site) to be cleaved when the CSTDPPC is internalized upon its CSTD binding to a cell-surface target. In some embodiments, the linker is a hydrolysable linker. In some embodiments, the linker is cleaved by proteolysis. In some embodiments, the linker includes a cleavage site for a protease. The linker can include a cleavage site for any suitable protease. Suitable proteases include, without limitation, cathepsin. In some embodiments, the linker includes a valine-citrulline linker or a glutamic acid-valine-citrulline linker. In some embodiments, the linker is a P-glucuronide linker. In some embodiments, the linker is pH sensitive. In some embodiments, the linker is sensitive to reducing conditions. Any suitable reducible linker can be used. Suitable reducible linkers include, without limitation, disulfide linkers. In some embodiments, the linker is photolabile. Any suitable photolabile linker can be used.

[0135] In some embodiments, the payload-to-cell-surface-targeting domain (“PCSTD”) ratios comprise a range between 1-400. In some embodiments, the PCSTD ratio is more than 1, for example 5, 10, or 20 or more to 400. In some embodiments, the ratios comprise 5-400, 5-395, 5-390, 5-385, 5-380, 5-375, 5-370, 5-365, 5-360, 5-355, 5-350, 5-345, -340, 5-335, 5-330, 5-325, 5-320, 5-315, 5-310, 5-305, 5-300, 5-295, 5-290, 5-285, 5-280, 5-75, 5-270, 5-265, 5-260, 5-255, 5-250, 5-245, 5-240, 5-235, 5-230, 5-225, 5-220, 5-215, 5-10, 5-205, 5-200, 5-195, 5-190, 5-185, 5-180, 5-175, 5-170, 5-165, 5-160, 5-155, 5-150, 5-45, 5-140, 5-135, 5-130, 5-125, 5-120, 5-115, 5-110, 5-105, 5-100 5-95, 5-90, 5-85, 5-80, 5-5, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-400, 10-95, 10-390, 10-385, 10-380, 10-375, 10-370, 10-365, 10-360, 10-355, 10-350, 10-345, 10-40, 10-335, 10-330, 10-325, 10-320, 10-315, 10-310, 10-305, 10-300, 10-295, 10-290, 10-85, 10-280, 10-275, 10-270, 10-265, 10-260, 10-255, 10-250, 10-245, 10-240, 10-235, 10-30, 10-225, 10-220, 10-215, 10-210, 10-205, 10-200, 10-195, 10-190, 10-185, 10-180, 10-75, 10-170, 10-165, 10-160, 10-155, 10-150, 10-145, 10-140, 10-135, 10-130, 10-125, 10-20, 10-115, 10-110, 10-105, 10-100 10-95, 10-90, 10-85, 10-80, 10-75, 10-70, 10-65, 10-60,0-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-400, 15-395, 15-390, 15-85, 15-380, 15-375, 15-370, 15-365, 15-360, 15-355, 15-350, 15-345, 15-340, 15-335, 15-30, 15-325, 15-320, 15-315, 15-310, 15-305, 15-300, 15-295, 15-290, 15-285, 15-280, 15-75, 15-270, 15-265, 15-260, 15-255, 15-250, 15-245, 15-240, 15-235, 15-230, 15-225, 15-20, 15-215, 15-210, 15-205, 15-200, 15-195, 15-190, 15-185, 15-180, 15-175, 15-170, 15-65, 15-160, 15-155, 15-150, 15-145, 15-140, 15-135, 15-130, 15-125, 15-120, 15-115, 15-10, 15-105, 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50,5-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-400, 20-395, 20-390, 20-385, 20-380, 20-375,0-370, 20-360, 20-355, 20-350, 20-345, 20-340, 20-335, 20-330, 20-325, 20-320, 20-315, 20-10, 20-305, 20-300, 20-295, 20-290, 20-285, 20-280, 20-275, 20-270, 20-265, 20-260, 20-55, 20-250, 20-245, 20-240, 20-235, 20-230, 20-225, 20-220, 20-215, 20-210, 20-205, 20-00, 20-195, 20-190, 20-185, 20-180, 20-175, 20-170, 20-165, 20-160, 20-155, 20-150, 20-45, 20-140, 20-135, 20-130, 20-125, 20-120, 20-115, 20-110, 20-105, 20-100, 20-95, 20-90,0-85, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25,5-400, 25-395, 25-390, 25-385, 25-380, 25-375, 25-370, 25-360, 25-355, 25-350, 25-345, 25-40, 25-335, 25-330, 25-325, 25-320, 25-315, 25-310, 25-305, 25-300, 25-295, 25-290, 25-85, 25-280, 25-275, 25-270, 25-265, 25-260, 25-255, 25-250, 25-245, 25-240, 25-235, 25-30, 25-225, 25-220, 25-215, 25-210, 25-205, 25-200, 25-195, 25-190, 25-185, 25-180, 25-75, 25-170, 25-165, 25-160, 25-155, 25-150, 25-145, 25-140, 25-135, 25-130, 25-125, 25-20, 25-115, 25-110, 25-105, 25-100, 25-95, 25-90, 25-85, 25-80, 25-75, 25-70, 25-65, 25-60, -55, 25-50, 25-45, 25-40, 25-35, 25-30, 30-400, 30-395, 30-390, 30-385, 30-380, 30-375,-370, 30-360, 30-355, 30-350, 30-345, 30-340, 30-335, 30-330, 30-325, 30-320, 30-315, 30-0, 30-305, 30-300, 30-295, 30-290, 30-285, 30-280, 30-275, 30-270, 30-265, 30-260, SO-5, 30-250, 30-245, 30-240, 30-235, 30-230, 30-225, 30-220, 30-215, 30-210, 30-205, 30-0, 30-195, 30-190, 30-185, 30-180, 30-175, 30-170, 30-165, 30-160, 30-155, 30-150, SO-5, 30-140, 30-135, 30-130, 30-125, 30-120, 30-115, 30-110, 30-105, 30-100, 30-95, 30-90,-85, 30-80, 30-75, 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-400, 35-395,-390, 35-385, 35-380, 35-375, 35-370, 35-360, 35-355, 35-350, 35-345, 35-340, 35-335, 35-0, 35-325, 35-320, 35-315, 35-310, 35-305, 35-300, 35-295, 35-290, 35-285, 35-280, 35-5, 35-270, 35-265, 35-260, 35-255, 35-250, 35-245, 35-240, 35-235, 35-230, 35-225, 35-0, 35-215, 35-210, 35-205, 35-200, 35-195, 35-190, 35-185, 35-180, 35-175, 35-170, 35-5, 35-160, 35-155, 35-150, 35-145, 35-140, 35-135, 35-130, 35-125, 35-120, 35-115, 35-0, 35-105, 35-100, 35-95, 35-90, 35-85, 35-80, 35-75, 35-70, 35-65, 35-60, 35-55, 35-50,-45, 35-40, 40-400, 40-395, 40-390, 40-385, 40-380, 40-375, 40-370, 40-360, 40-355, 40-0, 40-345, 40-340, 40-335, 40-330, 40-325, 40-320, 40-315, 40-310, 40-305, 40-300, 40-5, 40-290, 40-285, 40-280, 40-275, 40-270, 40-265, 40-260, 40-255, 40-250, 40-245, 40-0, 40-235, 40-230, 40-225, 40-220, 40-215, 40-210, 40-205, 40-200, 40-195, 40-190, 40-5, 40-180, 40-175, 40-170, 40-165, 40-160, 40-155, 40-150, 40-145, 40-140, 40-135, 40-0, 40-125, 40-120, 40-115, 40-110, 40-105, 40-100, 40-95, 40-90, 40-85, 40-80, 40-75, 40-, 40-65, 40-60, 40-55, 40-50, 40-45, 45-400, 45-395, 45-390, 45-385, 45-380, 45-375, 45-0, 45-360, 45-355, 45-350, 45-345, 45-340, 45-335, 45-330, 45-325, 45-320, 45-315, 45-0, 45-305, 45-300, 45-295, 45-290, 45-285, 45-280, 45-275, 45-270, 45-265, 45-260, 45-5, 45-250, 45-245, 45-240, 45-235, 45-230, 45-225, 45-220, 45-215, 45-210, 45-205, 45-0, 45-195, 45-190, 45-185, 45-180, 45-175, 45-170, 45-165, 45-160, 45-155, 45-150, 45-5, 45-140, 45-135, 45-130, 45-125, 45-120, 45-115, 45-110, 45-100, 45-105, 45-95, 45-90,-85, 45-80, 45-75, 45-70, 45-65, 45-60, 45-55, 45-50, 50-400, 50-395, 50-390, 50-385, 50-0, 50-375, 50-370, 50-360, 50-355, 50-350, 50-345, 50-340, 50-335, 50-330, 50-325, 50-0, 50-315, 50-310, 50-305, 50-300, 50-295, 50-290, 50-285, 50-280, 50-275, 50-270, 50-5, 50-260, 50-255, 50-250, 50-245, 50-240, 50-235, 50-230, 50-225, 50-220, 50-215, 50-0, 50-205, 50-200, 50-195, 50-190, 50-185, 50-180, 50-175, 50-170, 50-165, 50-160, SO-5, 50-150, 50-145, 50-140, 50-135, 50-130, 50-125, 50-120, 50-115, 50-110, 50-105, 50- 0, 50-95, 50-90, 50-85, 50-80, 50-75, 50-70, 50-65, 50-60, 50-55, 55-400, 55-395, 55-390,-385, 55-380, 55-375, 55-370, 55-360, 55-355, 55-350, 55-345, 55-340, 55-335, 55-330, 55-5, 55-320, 55-315, 55-310, 55-305, 55-300, 55-295, 55-290, 55-285, 55-280, 55-275, 55-0, 55-265, 55-260, 55-255, 55-250, 55-245, 55-240, 55-235, 55-230, 55-225, 55-220, 55-5, 55-210, 55-205, 55-200, 55-195, 55-190, 55-185, 55-180, 55-175, 55-170, 55-165, 55-0, 55-155, 55-150, 55-145, 55-140, 55-135, 55-130, 55-125, 55-120, 55-115, 55-110, 55-5, 55-100, 55-95, 55-90, 55-85, 55-80, 55-75, 55-70, 55-65, 55-60, 60-400, 60-395, 60-390,-385, 60-380, 60-375, 60-370, 60-360, 60-355, 60-350, 60-345, 60-340, 60-335, 60-330, 60-5, 60-320, 60-315, 60-310, 60-305, 60-300, 60-295, 60-290, 60-285, 60-280, 60-275, 60-0, 60-265, 60-260, 60-255, 60-250, 60-245, 60-240, 60-235, 60-230, 60-225, 60-220, 60-5, 60-210, 60-205, 60-200, 60-195, 60-190, 60-185, 60-180, 60-175, 60-170, 60-165, 60-0, 60-155, 60-150, 60-145, 60-140, 60-135, 60-130, 60-125, 60-120, 60-115, 60-110, 60-5, 60-100, 60-95, 60-90, 60-85, 60-80, 60-75, 60-70, 60-65, 65-400, 65-395, 65-390, 65-5, 65-380, 65-375, 65-370, 65-360, 65-355, 65-350, 65-345, 65-340, 65-335, 65-330, 65-5, 65-320, 65-315, 65-310, 65-305, 65-300, 65-295, 65-290, 65-285, 65-280, 65-275, 65-0, 65-265, 65-260, 65-255, 65-250, 65-245, 65-240, 65-235, 65-230, 65-225, 65-220, 65-5, 65-210, 65-205, 65-200, 65-195, 65-190, 65-185, 65-180, 65-175, 65-170, 65-165, 65-0, 65-155, 65-150, 65-145, 65-140, 65-135, 65-130, 65-125, 65-120, 65-115, 65-110, 65-5, 100-65 65-95, 65-90, 65-85, 65-80, 65-75, 65-70, 70-400, 70-395, 70-390, 70-385, 70-0, 70-375, 70-370, 70-360, 70-355, 70-350, 70-345, 70-340, 70-335, 70-330, 70-325, 70-0, 70-315, 70-310, 70-305, 70-300, 70-295, 70-290, 70-285, 70-280, 70-275, 70-270, 70-5, 70-260, 70-255, 70-250, 70-245, 70-240, 70-235, 70-230, 70-225, 70-220, 70-215, 70-0, 70-205, 70-200, 70-195, 70-190, 70-185, 70-180, 70-175, 70-170, 70-165, 70-160, 70-5, 70-150, 70-145, 70-140, 70-135, 70-130, 70-125, 70-120, 70-115, 70-110, 100-7070-105,-95, 70-90, 70-85, 70-80, 70-75, 75-400, 75-395, 75-390, 75-385, 75-380, 75-375, 75-370,-360, 75-355, 75-350, 75-345, 75-340, 75-335, 75-330, 75-325, 75-320, 75-315, 75-310, 75-5, 75-300, 75-295, 75-290, 75-285, 75-280, 75-275, 75-270, 75-265, 75-260, 75-255, 75-0, 75-245, 75-240, 75-235, 75-230, 75-225, 75-220, 75-215, 75-210, 75-205, 75-200, 75-5, 75-190, 75-185, 75-180, 75-175, 75-170, 75-165, 75-160, 75-155, 75-150, 75-145, 75-0, 75-135, 75-130, 75-125, 75-120, 75-115, 75-110, 75-100, 75-105, 75-95, 75-90, 75-85,-80, 80-400, 80-395, 80-390, 80-385, 80-380, 80-375, 80-370, 80-360, 80-355, 80-350, 80- 5, 80-340, 80-335, 80-330, 80-325, 80-320, 80-315, 80-310, 80-305, 80-300, 80-295, 80-0, 80-285, 80-280, 80-275, 80-270, 80-265, 80-260, 80-255, 80-250, 80-245, 80-240, SO-5, 80-230, 80-225, 80-220, 80-215, 80-210, 80-205, 80-200, 80-195, 80-190, 80-185, 80-0, 80-175, 80-170, 80-165, 80-160, 80-155, 80-150, 80-145, 80-140, 80-135, 80-130, SO-5, 80-120, 80-115, 80-110, 80-105, 100-85, 80-95, 80-90, 80-85, 85-400, 85-395, 85-390,-385, 85-380, 85-375, 85-370, 85-360, 85-355, 85-350, 85-345, 85-340, 85-335, 85-330, 85-5, 85-320, 85-315, 85-310, 85-305, 85-300, 85-295, 85-290, 85-285, 85-280, 85-275, 85-0, 85-265, 85-260, 85-255, 85-250, 85-245, 85-240, 85-235, 85-230, 85-225, 85-220, 85-5, 85-210, 85-205, 85-200, 85-195, 85-190, 85-185, 85-180, 85-175, 85-170, 85-165, 85-0, 85-155, 85-150, 85-145, 85-140, 85-135, 85-130, 85-125, 85-120, 85-115, 85-110, 85-5, 85-100, 85-95, 85-90, 90-400, 90-395, 90-390, 90-385, 90-380, 90-375, 90-370, 90-360,-355, 90-350, 90-345, 90-340, 90-335, 90-330, 90-325, 90-320, 90-315, 90-310, 90-305, 90-0, 90-295, 90-290, 90-285, 90-280, 90-275, 90-270, 90-265, 90-260, 90-255, 90-250, 90-5, 90-240, 90-235, 90-230, 90-225, 90-220, 90-215, 90-210, 90-205, 90-200, 90-195, 90-0, 90-185, 90-180, 90-175, 90-170, 90-165, 90-160, 90-155, 90-150, 90-145, 90-140, 90-5, 90-130, 90-125, 90-120, 90-115, 90-110, 90-100, 90-105, 90-95, 95-400, 95-395, 95-390,-385, 95-380, 95-375, 95-370, 95-360, 95-355, 95-350, 95-345, 95-340, 95-335, 95-330, 95-5, 95-320, 95-315, 95-310, 95-305, 95-300, 95-295, 95-290, 95-285, 95-280, 95-275, 95-0, 95-265, 95-260, 95-255, 95-250, 95-245, 95-240, 95-235, 95-230, 95-225, 95-220, 95-5, 95-210, 95-205, 95-200, 95-195, 95-190, 95-185, 95-180, 95-175, 95-170, 95-165, 95-0, 95-155, 95-150, 95-145, 95-140, 95-135, 95-130, 95-125, 95-120, 95-115, 95-110, 95-5, 95-100, 100-400, 100-395, 100-390, 100-385, 100-380, 100-375, 100-370, 100-360, 1 GO-5, 100-350, 100-345, 100-340, 100-335, 100-330, 100-325, 100-320, 100-315, 100-310,0-305, 100-300, 100-295, 100-290, 100-285, 100-280, 100-275, 100-270, 100-265, 100-0, 100-255, 100-250, 100-245, 100-240, 100-235, 100-230, 100-225, 100-220, 100-215,0-210, 100-205, 100-200, 100-195, 100-190, 100-185, 100-180, 100-175, 100-170, 100-5, 100-160, 100-155, 100-150, 100-145, 100-140, 100-135, 100-130, 100-125, 100-120,0-115, 100-110, 100-105, 105-400, 105-395, 105-390, 105-385, 105-380, 105-375, 105-0, 105-360, 105-355, 105-350, 105-345, 105-340, 105-335, 105-330, 105-325, 105-320,5-315, 105-310, 105-305, 105-300, 105-295, 105-290, 105-285, 105-280, 105-275, 105-0, 105-265, 105-260, 105-255, 105-250, 105-245, 105-240, 105-235, 105-230, 105-225, -220, 105-215, 105-210, 105-205, 105-200, 105-195, 105-190, 105-185, 105-180, 105-, 105-170, 105-165, 105-160, 105-155, 105-150, 105-145, 105-140, 105-135, 105-130,-125, 105-120, 105-115, 105-110, 110-400, 110-395, 110-390, 110-385, 110-380, 110-, 110-370, 110-360, 110-355, 110-350, 110-345, 110-340, 110-335, 110-330, 110-325,-320, 110-315, 110-310, 110-305, 110-300, 110-295, 110-290, 110-285, 110-280, 110-, 110-270, 110-265, 110-260, 110-255, 110-250, 110-245, 110-240, 110-235, 110-230,-225, 110-220, 110-215, 110-210, 110-205, 110-200, 110-195, 110-190, 110-185, 110-, 110-175, 110-170, 110-165, 110-160, 110-155, 110-150, 110-145, 110-140, 110-135,-130, 110-125, 110-120, 110-115, 115-400, 115-395, 115-390, 115-385, 115-380, 115-, 115-370, 115-360, 115-355, 115-350, 115-345, 115-340, 115-335, 115-330, 115-325,-320, 115-315, 115-310, 115-305, 115-300, 115-295, 115-290, 115-285, 115-280, 115-, 115-270, 115-265, 115-260, 115-255, 115-250, 115-245, 115-240, 115-235, 115-230,-225, 115-220, 115-215, 115-210, 115-205, 115-200, 115-195, 115-190, 115-185, 115-, 115-175, 115-170, 115-165, 115-160, 115-155, 115-150, 115-145, 115-140, 115-135,-130, 115-125, 115-120, 120-400, 120-395, 120-390, 120-385, 120-380, 120-375, 120-, 120-360, 120-355, 120-350, 120-345, 120-340, 120-335, 120-330, 120-325, 120-320,-315, 120-310, 120-305, 120-300, 120-295, 120-290, 120-285, 120-280, 120-275, 120-, 120-265, 120-260, 120-255, 120-250, 120-245, 120-240, 120-235, 120-230, 120-225,-220, 120-215, 120-210, 120-205, 120-200, 120-195, 120-190, 120-185, 120-180, 120-, 120-170, 120-165, 120-160, 120-155, 120-150, 120-145, 120-140, 120-135, 120-130,-125, 125-400, 125-395, 125-390, 125-385, 125-380, 125-375, 125-370, 125-360, 125-, 125-350, 125-345, 125-340, 125-335, 125-330, 125-325, 125-320, 125-315, 125-310,-305, 125-300, 125-295, 125-290, 125-285, 125-280, 125-275, 125-270, 125-265, 125-, 125-255, 125-250, 125-245, 125-240, 125-235, 125-230, 125-225, 125-220, 125-215,-210, 125-205, 125-200, 125-195, 125-190, 125-185, 125-180, 125-175, 125-170, 125-, 125-160, 125-155, 125-150, 125-145, 125-140, 125-135, 125-130, 130-400, 130-395,-390, 130-385, 130-380, 130-375, 130-370, 130-360, 130-355, 130-350, 130-345, 130-, 130-335, 130-330, 130-325, 130-320, 130-315, 130-310, 130-305, 130-300, 130-295,-290, 130-285, 130-280, 130-275, 130-270, 130-265, 130-260, 130-255, 130-250, 130-, 130-240, 130-235, 130-230, 130-225, 130-220, 130-215, 130-210, 130-205, 130-200,-195, 130-190, 130-185, 130-180, 130-175, 130-170, 130-165, 130-160, 130-155, 130- 150, 130-145, 130-140, 130-135, 135-400, 135-395, 135-390, 135-385, 135-380, 135-375, 135-370, 135-360, 135-355, 135-350, 135-345, 135-340, 135-335, 135-330, 135-325, 135- 20, 135-315, 135-310, 135-305, 135-300, 135-295, 135-290, 135-285, 135-280, 135-275, 135-270, 135-265, 135-260, 135-255, 135-250, 135-245, 135-240, 135-235, 135-230, 135- 25, 135-220, 135-215, 135-210, 135-205, 135-200, 135-195, 135-190, 135-185, 135-180, 135-175, 135-170, 135-165, 135-160, 135-155, 135-150, 135-145, 135-140, 140-400, MO- 95, 140-390, 140-385, 140-380, 140-375, 140-370, 140-360, 140-355, 140-350, 140-345, 140-340, 140-335, 140-330, 140-325, 140-320, 140-315, 140-310, 140-305, 140-300, MO- 95, 140-290, 140-285, 140-280, 140-275, 140-270, 140-265, 140-260, 140-255, 140-250, 140-245, 140-240, 140-235, 140-230, 140-225, 140-220, 140-215, 140-210, 140-205, 140- 00, 140-195, 140-190, 140-185, 140-180, 140-175, 140-170, 140-165, 140-160, 140-155, 140-150, 140-145, 145-400, 145-395, 145-390, 145-385, 145-380, 145-375, 145-370, 145- 60, 145-355, 145-350, 145-345, 145-340, 145-335, 145-330, 145-325, 145-320, 145-315, 145-310, 145-305, 145-300, 145-295, 145-290, 145-285, 145-280, 145-275, 145-270, MS- 65, 145-260, 145-255, 145-250, 145-245, 145-240, 145-235, 145-230, 145-225, 145-220, 145-215, 145-210, 145-205, 145-200, 145-195, 145-190, 145-185, 145-180, 145-175, MS- MO, 145-165, 145-160, 145-155, 145-150, 150-400, 150-395, 150-390, 150-385, 150-380, 150-375, 150-370, 150-360, 150-355, 150-350, 150-345, 150-340, 150-335, 150-330, ISO- 25, 150-320, 150-315, 150-310, 150-305, 150-300, 150-295, 150-290, 150-285, 150-280, 150-275, 150-270, 150-265, 150-260, 150-255, 150-250, 150-245, 150-240, 150-235, 150- 30, 150-225, 150-220, 150-215, 150-210, 150-205, 150-200, 150-195, 150-190, 150-185, 150-180, 150-175, 150-170, 150-165, 150-160, 150-155, 155-400, 155-395, 155-390, 155- 85, 155-380, 155-375, 155-370, 155-360, 155-355, 155-350, 155-345, 155-340, 155-335, 155-330, 155-325, 155-320, 155-315, 155-310, 155-305, 155-300, 155-295, 155-290, 155- 85, 155-280, 155-275, 155-270, 155-265, 155-260, 155-255, 155-250, 155-245, 155-240, 155-235, 155-230, 155-225, 155-220, 155-215, 155-210, 155-205, 155-200, 155-195, 155- 190, 155-185, 155-180, 155-175, 155-170, 155-165, 155-160, 160-400, 160-395, 160-390, 160-385, 160-380, 160-375, 160-370, 160-360, 160-355, 160-350, 160-345, 160-340, 160- 35, 160-330, 160-325, 160-320, 160-315, 160-310, 160-305, 160-300, 160-295, 160-290, 160-285, 160-280, 160-275, 160-270, 160-265, 160-260, 160-255, 160-250, 160-245, 160- 40, 160-235, 160-230, 160-225, 160-220, 160-215, 160-210, 160-205, 160-200, 160-195, -190, 160-185, 160-180, 160-175, 160-170, 160-165, 165-400, 165-395, 165-390, 165-, 165-380, 165-375, 165-370, 165-360, 165-355, 165-350, 165-345, 165-340, 165-335,-330, 165-325, 165-320, 165-315, 165-310, 165-305, 165-300, 165-295, 165-290, 165-, 165-280, 165-275, 165-270, 165-265, 165-260, 165-255, 165-250, 165-245, 165-240,-235, 165-230, 165-225, 165-220, 165-215, 165-210, 165-205, 165-200, 165-195, 165-, 165-185, 165-180, 165-175, 165-170, 170-400, 170-395, 170-390, 170-385, 170-380,-375, 170-370, 170-360, 170-355, 170-350, 170-345, 170-340, 170-335, 170-330, 170-, 170-320, 170-315, 170-310, 170-305, 170-300, 170-295, 170-290, 170-285, 170-280,-275, 170-270, 170-265, 170-260, 170-255, 170-250, 170-245, 170-240, 170-235, 170-, 170-225, 170-220, 170-215, 170-210, 170-205, 170-200, 170-195, 170-190, 170-185,-180, 170-175, 175-400, 175-395, 175-390, 175-385, 175-380, 175-375, 175-370, 175-, 175-360, 175-355, 175-350, 175-345, 175-340, 175-335, 175-330, 175-325, 175-320,-315, 175-310, 175-305, 175-300, 175-295, 175-290, 175-285, 175-280, 175-275, 175-, 175-265, 175-260, 175-255, 175-250, 175-245, 175-240, 175-235, 175-230, 175-225,-220, 175-215, 175-210, 175-205, 175-200, 175-195, 175-190, 175-185, 175-180, 180-, 180-395, 180-390, 180-385, 180-380, 180-375, 180-365, 180-370, 180-360, 180-355,-350, 180-345, 180-340, 180-335, 180-330, 180-325, 180-320, 180-315, 180-310, 180-, 180-300, 180-295, 180-290, 180-285, 180-280, 180-275, 180-270, 180-265, 180-260,-255, 180-250, 180-245, 180-240, 180-235, 180-230, 180-225, 180-220, 180-215, 180-, 180-205, 180-200, 180-195, 180-190, 180-185, 185-400, 185-395, 185-390, 185-385,-380, 185-375, 185-370, 185-365, 185-360, 185-355, 185-350, 185-345, 185-340, 185-, 185-330, 185-325, 185-320, 185-315, 185-310, 185-305, 185-300, 185-295, 185-290,-285, 185-280, 185-275, 185-270, 185-265, 185-260, 185-255, 185-250, 185-245, 185-, 185-235, 185-230, 185-225, 185-220, 185-215, 185-210, 185-205, 185-200, 185-195,-190, 190-400, 190-395, 190-390, 190-385, 190-380, 190-375, 190-370, 190-365, 190-, 190-355, 190-350, 190-345, 190-340, 190-335, 190-330, 190-325, 190-320, 190-315,-310, 190-305, 190-300, 190-295, 190-290, 190-285, 190-280, 190-275, 190-270, 190-, 190-260, 190-255, 190-250, 190-245, 190-240, 190-235, 190-230, 190-225, 190-220,-215, 190-210, 190-205, 190-200, 190-195, 195-400, 195-395, 195-390, 195-385, 195-, 195-375, 195-370, 195-365, 195-360, 195-355, 195-350, 195-345, 195-340, 195-335,-330, 195-325, 195-320, 195-315, 195-310, 195-305, 195-300, 195-295, 195-290, 195- , 195-280, 195-275, 195-270, 195-265, 195-260, 195-255, 195-250, 195-245, 195-240,-235, 195-230, 195-225, 195-220, 195-215, 195-210, 195-205, 195-200, 200-400, 200-, 200-390, 200-385, 200-380, 200-375, 200-370, 200-365, 200-360, 200-355, 200-350,-345, 200-340, 200-335, 200-330, 200-325, 200-320, 200-315, 200-310, 200-305, 200-, 200-295, 200-290, 200-285, 200-280, 200-275, 200-270, 200-265, 200-260, 200-255,-250, 200-245, 200-240, 200-235, 200-230, 200-225, 200-220, 200-215, 200-210, 200-, 205-400, 205-395, 205-390, 205-385, 205-380, 205-375, 205-370, 205-365, 205-360,-355, 205-350, 205-345, 205-340, 205-335, 205-330, 205-325, 205-320, 205-315, 205-, 205-305, 205-300, 205-295, 205-290, 205-285, 205-280, 205-275, 205-270, 205-265,-260, 205-255, 205-250, 205-245, 205-240, 205-235, 205-230, 205-225, 205-220, 205-, 205-210, 210-400, 210-395, 210-390, 210-385, 210-380, 210-375, 210-370, 210-365,-360, 210-355, 210-350, 210-345, 210-340, 210-335, 210-330, 210-325, 210-320, 210-, 210-310, 210-305, 210-300, 210-295, 210-290, 210-285, 210-280, 210-275, 210-270,-265, 210-260, 210-255, 210-250, 210-245, 210-240, 210-235, 210-230, 210-225, 210-, 210-215, 215-400, 215-395, 215-390, 215-385, 215-380, 215-375, 215-370, 215-365,-360, 215-355, 215-350, 215-345, 215-340, 215-335, 215-330, 215-325, 215-320, 215-, 215-310, 215-305, 215-300, 215-295, 215-290, 215-285, 215-280, 215-275, 215-270,-265, 215-260, 215-255, 215-250, 215-245, 215-240, 215-235, 215-230, 215-225, 215-, 220-400, 220-395, 220-390, 220-385, 220-380, 220-375, 220-370, 220-365, 220-360,-355, 220-350, 220-345, 220-340, 220-335, 220-330, 220-325, 220-320, 220-315, 220-, 220-305, 220-300, 220-295, 220-290, 220-285, 220-280, 220-275, 220-270, 220-265,-260, 220-255, 220-250, 220-245, 220-240, 220-235, 220-230, 220-225, 225-400, 225-, 225-390, 225-385, 225-380, 225-375, 225-370, 225-365, 225-360, 225-355, 225-350,-345, 225-340, 225-335, 225-330, 225-325, 225-320, 225-315, 225-310, 225-305, 225-, 225-295, 225-290, 225-285, 225-280, 225-275, 225-270, 225-265, 225-260, 225-255,-250, 225-245, 225-240, 225-235, 225-230, 230-400, 230-395, 230-390, 230-385, 230-, 230-375, 230-370, 230-365, 230-360, 230-355, 230-350, 230-345, 230-340, 230-335,-330, 230-325, 230-320, 230-315, 230-310, 230-305, 230-300, 230-295, 230-290, 230-, 230-280, 230-275, 230-270, 230-265, 230-260, 230-255, 230-250, 230-245, 230-240,-235, 235-400, 235-395, 235-390, 235-385, 235-380, 235-375, 235-370, 235-365, 235-, 235-355, 235-350, 235-345, 235-340, 235-335, 235-330, 235-325, 235-320, 235-315, -310, 235-305, 235-300, 235-295, 235-290, 235-285, 235-280, 235-275, 235-270, 235-, 235-260, 235-255, 235-250, 235-245, 235-240, 240-400, 240-395, 240-390, 240-385,-380, 240-375, 240-370, 240-365, 240-360, 240-355, 240-350, 240-345, 240-340, 240-, 240-330, 240-325, 240-320, 240-315, 240-310, 240-305, 240-300, 240-295, 240-290,-285, 240-280, 240-275, 240-270, 240-265, 240-260, 240-255, 240-250, 240-245, 245-, 245-395, 245-390, 245-385, 245-380, 245-375, 245-370, 245-365, 245-360, 245-355,-350, 245-345, 245-340, 245-335, 245-330, 245-325, 245-320, 245-315, 245-310, 245-, 245-300, 245-295, 245-290, 245-285, 245-280, 245-275, 245-270, 245-265, 245-260,-255, 245-250, 250-400, 250-395, 250-390, 250-385, 250-380, 250-375, 250-370, 250-, 250-360, 250-355, 250-350, 250-345, 250-340, 250-335, 250-330, 250-325, 250-320,-315, 250-310, 250-305, 250-300, 250-295, 250-290, 250-285, 250-280, 250-275, 250-, 250-265, 250-260, 250-255, 255-400, 255-395, 255-390, 255-385, 255-380, 255-375,-370, 255-365, 255-360, 255-355, 255-350, 255-345, 255-340, 255-335, 255-330, 255-, 255-320, 255-315, 255-310, 255-305, 255-300, 255-295, 255-290, 255-285, 255-280,-275, 255-270, 255-265, 255-260, 260-400, 260-395, 260-390, 260-385, 260-380, 260-, 260-370, 260-365, 260-360, 260-355, 260-350, 260-345, 260-340, 260-335, 260-330,-325, 260-320, 260-315, 260-310, 260-305, 260-300, 260-295, 260-290, 260-285, 260-, 260-275, 260-270, 260-265, 265-400, 265-395, 265-390, 265-385, 265-380, 265-375,-370, 265-365, 265-360, 265-355, 265-350, 265-345, 265-340, 265-335, 265-330, 265-, 265-320, 265-315, 265-310, 265-305, 265-300, 265-295, 265-290, 265-285, 265-280,-275, 265-270, 270-400, 270-395, 270-390, 270-385, 270-380, 270-375, 270-370, 270-, 270-365, 270-355, 270-350, 270-345, 270-340, 270-335, 270-330, 270-325, 270-320,-315, 270-310, 270-305, 270-300, 270-295, 270-290, 270-285, 270-280, 270-275, 275-, 275-395, 275-390, 275-385, 275-380, 275-375, 275-370, 275-365, 275-360, 275-355,-350, 275-345, 275-340, 275-335, 275-330, 275-325, 275-320, 275-315, 275-310, 275-, 275-300, 275-295, 275-290, 275-285, 275-280, 280-400, 280-395, 280-390, 280-385,-380, 280-375, 280-370, 280-365, 280-360, 280-355, 280-350, 280-345, 280-340, 280-, 280-330, 280-325, 280-320, 280-315, 280-310, 280-305, 280-300, 280-295, 280-290,-285, 285-400, 285-395, 285-390, 285-385, 285-380, 285-375, 285-370, 285-365, 285-, 285-355, 285-350, 285-345, 285-340, 285-335, 285-330, 285-325, 285-320, 285-315,-310, 285-305, 285-300, 285-295, 285-290, 290-400, 290-395, 290-390, 290-385, 290- 380, 290-375, 290-370, 290-365, 290-360, 290-355, 290-350, 290-345, 290-340, 290-335, 290-330, 290-325, 290-320, 290-315, 290-310, 290-305, 290-300, 290-295, 295-400, 295- 395, 295-390, 295-385, 295-380, 295-375, 295-370, 295-365, 295-360, 295-355, 295-350, 295-345, 295-340, 295-335, 295-330, 295-325, 295-320, 295-315, 295-310, 295-305, 295- 300, 300-400, 300-395, 300-390, 300-385, 300-380, 300-375, 300-370, 300-365, 300-360, 300-355, 300-350, 300-345, 300-340, 300-335, 300-330, 300-325, 300-320, 300-315, 300- 310, 300-305, 305-400, 305-395, 305-390, 305-385, 305-380, 305-375, 305-370, 305-360, 305-360, 305-355, 305-350, 305-345, 305-340, 305-335, 305-330, 305-325, 305-320, 305- 315, 305-310, 310-400, 310-395, 310-390, 310-385, 310-380, 310-375, 310-370, 310-365, 310-360, 310-355, 310-350, 310-345, 310-340, 310-335, 310-330, 310-325, 310-320, 310- 315, 315-400, 315-395, 315-390, 315-385, 315-380, 315-375, 315-370, 315-365, 315-360, 315-355, 315-350, 315-345, 315-340, 315-335, 315-330, 315-325, 315-320, 320-400, 320- 395, 320-390, 320-385, 320-380, 320-375, 320-370, 320-360, 320-360, 320-355, 320-350, 320-345, 320-340, 320-335, 320-330, 320-325, 325-400, 325-395, 325-390, 325-385, 325- 380, 325-375, 325-370, 325-365, 325-360, 325-355, 325-350, 325-345, 325-340, 325-335, 325-330, 330-400, 330-395, 330-390, 330-385, 330-380, 330-375, 330-370, 330-360, 330- 355, 330-350, 330-345, 330-340, 330-335, 335-400, 335-395, 335-390, 335-385, 335-380, 335-375, 335-370, 335-365, 335-360, 335-355, 335-350, 335-345, 335-340, 340-400, 340- 395, 340-390, 340-385, 340-380, 340-375, 340-370, 340-365, 340-360, 340-355, 340-350, 340-345, 345-400, 345-395, 345-390, 345-385, 345-380, 345-375, 345-370, 345-365, 345- 360, 345-355, 345-350, 350-400, 350-395, 350-390, 350-385, 350-380, 350-375, 350-370, 350-365, 350-360, 350-355, 355-400, 355-395, 355-390, 355-385, 355-380, 355-375, 355- 370, 355-365, 355-360, 360-400, 360-395, 360-390, 360-385, 360-380, 360-375, 360-370, 370-400, 370-395, 370-390, 370-385, 370-380, 370-375, 375-400, 375-395, 375-390, 375- 385, 375-380, 380-400, 380-395, 380-390, 380-385, 390-400, 390-395, and 395-400 including any range above any one of the preceding values and any range between any two of the preceding values. In some embodiments, the PCSTD ratio comprises or is in the range of 5- 400. In some embodiments, the PCSTD ratio comprises or is in the range of 10-400. In some embodiments, the PCSTD ratio comprises or is in the range of 20-400. In some embodiments, the PCSTD ratio is in the range of 30-200. In some embodiments, the PCSTD ratio is in the range of 50-150. In some embodiments, the PCSTD ratio is in the range of 10-300. In some embodiments, the PCSTD ratio is at least 5. In some embodiments, the PCSTD ratio is at least 10. In some embodiments, the PCSTD ratio is at least 20. In some embodiments, the PCSTD ratio is at least 30. In some embodiments, the PCSTD ratio denotes the ratio for at least one of the payloads. In some embodiments, the PCSTD ratio denotes the ratio for two or more different payloads (e.g., an average ratio for two or more different payloads). In some embodiments, the PCSTD ratio denotes the ratio for all of the payloads (e.g., the ratio of all of the different payloads to cell-surface targeting domain).

[0136] In some embodiments, a high concentration of payload molecules localize inside and/or outside the targeted cells. In some embodiments, the targeted payload molecules comprise concentrations efficacious for purposes including but not limited to therapeutic, identification, localization, sorting, and/or isolation.

[0137] In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 300,000 and about 1,750,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 300,000 and about 1,750,000 Da, as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC- MALS”). In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 10,000 and about 1,750,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 10,000 and about 1,750,000 Da, as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC- MALS”). Optionally, the polymer (e.g., copolymer) has a molecular weight between about 500,000 and about 1,000,000 Da. Optionally, the polymer (e.g., copolymer) has a molecular weight of between about 600,000 to about 800,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) comprises 300,00 Da or greater. In some embodiments, the molecular weight of the polymer is 10,000, 20,000, 30,000, 40,000, 50,000 60,000, 70,000 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, or 300,000 to 1,750,000 Da, including any range there between and any range starting with the preceding values. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 10,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 25,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 38,000 Da. Tn some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 50,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 100,000Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 150,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 180,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 250,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 290,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) is or comprises 300,000 Da or greater.

[0138] In some embodiments, an antibody conjugate comprising an anti-VEGFR2 immunoglobulin G (IgG) bonded to a polymer (e.g., copolymer), which polymer comprises MPC monomers, optionally, the polymer has 9 arms; and the polymer has a molecular weight of between about 600,000 to about 800,000 Da. In some embodiments the polymer has a molecular weight of between 600,000 to about 800,000 Da.

[0139] In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 300,000 and 1,750,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 10,000 and 1,750,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 500,000 and 1,000,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight of between about 600,000 to 800,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 10,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 25,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 38,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 50,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 100,000Da. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 150,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 180,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 250,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 290,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) comprises 300,00 Da or greater. In some embodiment, the molecular weight of the polymer (e.g., copolymer) is, is about, or is at least 10,000, 20,000, 30,000, 40,000, 50,000 60,000, 70,000 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, or 300,000 to 1,750,000 Da, including any range there between and any range starting with the preceding values.

[0140] In some embodiments, the polymer (e.g., copolymer) conjugated to the antibody (or other protein) has a molecular weight between about 300,000 and about 1,750,000 Da as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC-MALS”). In some embodiments, the polymer (e.g., copolymer) conjugated to the antibody (or other protein) has a molecular weight between about 10,000 and about 1,750,000 Da as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC-MALS”). In some embodiments, the molecular weight of the polymer (e.g., copolymer) is or comprises 300,00 Da or greater. In some embodiment, the molecular weight of the polymer (e.g., copolymer) is 10,000, 20,000, 30,000, 40,000, 50,000 60,000, 70,000 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000 to 1,750,000 including any range there between and any range starting with the preceding values. In some embodiments, the polymer (e.g., copolymer) comprises a molecular weight of about 10,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 25,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 38,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 50,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 100,000Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 150,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 180,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 250,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 290,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) is or comprises 300,00 Da or greater. [0141] In some embodiments, the antibody (or other protein) is further conjugated to a polymer (e.g., copolymer) to form a bioconjugate, and wherein the bioconjugate has a molecular weight between about 450,000 and 1,900,000 Daltons.

[0142] Multi-angle light scattering (MALS) is a technique of analyzing macromolecules where the laser light impinges on the molecule, the oscillating electric field of the light induces an oscillating dipole within it. This oscillating dipole will re-radiate light and can be measured using a MALS detector such as Wyatt miniDawn TREOS. The intensity of the radiated light depends on the magnitude of the dipole induced in the macromolecule which in turn is proportional to the polarizability of the macromolecule, the larger the induced dipole, and hence, the greater the intensity of the scattered light. Therefore, in order to analyze the scattering from a solution of such macromolecules, one should know their polarizability relative to the surrounding medium (e g., the solvent). This may be determined from a measurement of the change, A//, of the solution's refractive index n with the molecular concentration change, Ac, by measuring the dn/dc (= n/ c) value using a Wyatt Optilab T- rEX differential refractometer. Two molar weight parameters that MALS determination employ are number average molecular weight (Mn) and weight average molecular weight (Mw) where the poly dispersity index (PDI) equals Mw divided by Mn. SEC also allows another average molecular weight determination of the peak molecular weight Mp which is defined as the molecular weight of the highest peak at the SEC.

[0143] In some embodiments, the polymer (e.g., copolymer) that is added has a molecular weight between about 300,000 and about 1,750,000 Da (SEC-MALs). In some embodiments, the polymer (e.g., copolymer) that is added has a molecular weight between about 10,000 and about 1,750,000 Da (SEC-MALs). In some embodiments, the polymer (e.g., copolymer) has a molecular weight between about 500,000 and about 1,000,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight of between about 600,000 to about 900,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight of between about 750,000 to about 850,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight of between about 800,000 to about 850,000 Da. In some embodiments, the polymer (e.g., copolymer) has a molecular weight of between about 750,000 to about 800,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) is or comprises 300,00 Da or greater. In some embodiment, the range can be 10,000, 20,000, 30,000, 40,000, 50,000 60,000, 70,000 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, or 300,000 to 1,750,000 including any range there between and any range starting with the preceding values. In some embodiments, the polymer (e.g., copolymer) comprises or has a molecular weight of about 10,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 25,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 38,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises or has a molecular weight of about 50,000 Da. In some embodiments, the polymer (e.g., copolymer) comprises r has a molecular weight of about 100,000Da. In some embodiments, the polymer (e.g., copolymer) comprises r has a molecular weight of about 150,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 180,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 290,000 Da. In some embodiments, the polymer (e.g., copolymer) has or comprises a molecular weight of about 250,000 Da. In some embodiments, the molecular weight of the polymer (e.g., copolymer) is or comprises 300,000 Da or greater.

[0144] In some embodiments, any of the antibodies (or proteins) described herein can be further conjugated to a polymer (e.g., copolymer) to form a bioconjugate. The molecular weight of the bioconjugate (in total, SEC-MALs) can be between about 350,000 and 2,000,000 Daltons, for example, between about 450,000 and 1,900,000 Daltons, between about 550,000 and 1,800,000 Daltons, between about 650,000 and 1,700,000 Daltons, between about 750,000 and 1,600,000 Daltons, between about 850,000 and 1,500,000 Daltons, between about 900,000 and 1,400,000 Daltons, between about 950,000 and 1,300,000 Daltons, between about 900,000 and 1,000,000 Daltons, between about 1,000,000 and 1,300,000 Daltons, between about 850,000 and 1,300,000 Daltons, between about 850,000 and 1,000,000 Daltons, and between about 1,000,000 and 1,200,000 Daltons. In some embodiments, the polymer (e.g., copolymer) has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.

[0145] In some embodiments, polymer-antibody (or other protein) conjugates have a polymer (e.g., copolymer) portion with a molecular weight of between 100,000 and 1,500,000 Da. Tn some embodiments, the conjugate has a polymer (e.g., copolymer) portion with a molecular weight between 500,000 and 1,000,000 Da. In some embodiments, the conjugate has a polymer (e.g., copolymer) portion with a molecular weight between 600,000 to 800,000 Da. In some embodiments, the conjugate has a polymer (e.g., copolymer) portion with a molecular weight between 600,000 and 850,000 Da and has 9 arms. When a molecular weight is given for an antibody (or other protein) conjugated to a polymer(e.g., copolymer), the molecular weight will be the addition of the molecular weight of the protein, including any carbohydrate moi eties associated therewith, and the molecular weight of the polymer (e.g., copolymer).

[0146] In some embodiments, the polymer-antibody (or other protein) conjugate has a HEMA-PC polymer which has a molecular weight measured by Mw of between about 100 kDa and 1650 kDa is provided. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 500 kDa and 1000 kDa. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 600 kDa to about 900 kDa. In some embodiments, the polymer molecular weight as measured by Mw is 750 kDa plus or minus 15%.

[0147] In some embodiments, the MPC polymer has a molecular weight between about 300,000 and 1,750,000 Da. In some embodiments, the MPC polymer has a molecular weight between about 10,000 and 1,750,000 Da. In some embodiments, the MPC polymer has a molecular weight between about 500,000 and 1,000,000 Da or between about 600,000 to 900,000 Da. In some embodiment the range can be 10,000, 20,000, 30,000, 40,000, 50,000 60,000, 70,000 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000 to 1,750,000 including any range there between and any range starting with the preceding values. In some embodiments, the polymer comprises a molecular weight of about 10,000 Da. In some embodiments, the polymer comprises a molecular weight of about 50,000 Da. In some embodiments, the polymer comprises a molecular weight of about 100,000Da. In some embodiments, the polymer comprises a molecular weight of about 150,000 Da. In some embodiments, the polymer comprises a molecular weight of about 250,000 Da. In some embodiments, the molecular weight of the polymer comprises 300,00 Da or greater. [0148] In some embodiments, the cell surface targeting domain polymer payload conjugate (CSTDPPC) comprises a payload wherein the payload comprises oligonucleotides. In some embodiments, the payload comprises a small molecule. In some embodiments, the payload comprises a peptide. In some embodiments, the payload comprises an aptamer. In some embodiments, the payload comprises a macrocycle. In some embodiments, the payload comprises a cyclic peptide. In some embodiments, the payload comprises a protein. In some embodiments, the payload comprises a tRNA. In some embodiments, the payload comprises proteins and small molecules. In some embodiments, the payload comprises proteins and peptides. In some embodiments, the payload comprises proteins and aptamers, In some embodiments, the payload comprises proteins and macrocycles. In some embodiments, the payload comprises proteins and cyclic peptides. In some embodiments, the payload comprises oligonucleotides and small molecules. In some embodiments, the payload comprises oligonucleotides and peptides. In some embodiments, the payload comprises oligonucleotides and aptamers. In some embodiments, the payload comprises oligonucleotides and macrocycles. In some embodiments, the payload comprises oligonucleotides and cyclic peptides. In some embodiments, the payload comprises small molecules and peptides. In some embodiments, the payload comprises small molecules and aptamers. In some embodiments, the payload comprises small molecules and macrocycles. In some embodiments, the payload comprises small molecules and cyclic peptides. In some embodiments, the payload comprises peptides and aptamers. In some embodiments, the payload comprises peptides and macrocycles. In some embodiments, the payload comprises peptides and cyclic peptides. In some embodiments, the payload comprises aptamers and macrocycles. In some embodiments, the payload comprises aptamers and cyclic peptides. In some embodiments, the payload comprises macrocycles and cyclic peptides. In some embodiments, the payload comprises of a combination of two or more oligonucleotides, small molecules, aptamers, cyclic peptide, macrocycles and/or proteins. A non-limiting example would comprise a combination of aptamers and peptides and/or small molecules. In some embodiments, the payload comprises a combination of two or more different molecules of a specific type of payload molecule with non-limiting examples comprising combinations of two or more different small molecules and two or more different peptides. [0149] In some embodiments, KSI-301 can be employed in any of the present embodiments or arrangements. In some embodiments, the antibody comprises a heavy chain amino acid variable region that comprises SEQ ID NO: 1 and a light chain amino acid variable region that comprises SEQ ID NO: 2 (FIG.9). In some embodiments, the antibody is conjugated to one or more of the polymers provided herein. In some embodiments, the conjugated antibody is, is at least, or is about 90, 92, 94, 95, 96, 97, 98, 99% identical, or is about 100%> identical, to SEQ ID NO: 1 and/or 2. In some embodiments, the antibody contains the 6 CDRs within SEQ ID NO: 1 and SEQ ID NO: 2, as well as a point mutation of L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the conjugated antibody is, is at least, or is about 90, 92, 94, 95, 96, 97, 98, 99% identical, or is about 100% identical, to SEQ ID NO: 1 and/or 2 and includes the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering). In some embodiments, an antibody that binds to VEGF-A is provided. The antibody comprises: a CDRHI that is the CDRHI in SEQ ID NO: 1, a CDRH2 that is the CDRH2 in SEQ ID NO: 1, a CDRH3 that is the CDRH3 in SEQ ID NO: 1, a CDRLI that is the CDRLI in SEQ ID NO: 2, a CDRL2 that is the CDRL2 in SEQ ID NO: 2, a CDRL3 that is the CDRL3 in SEQ ID NO: 2, at least one of the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering). In some embodiments, the VEGF antibody has the amino acid sequences shown in FIG. 9.

References

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Yurkovetskiy AV, Yin M, Bodyak N, Stevenson CA, Thomas JD, Hammond CE, Qin L, Zhu B, Gumerov DR, Ter-Ovanesyan E, et al. 2015. A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy. Cancer. Res. 75: 3365- 3372. [0150] Additional, non-limiting embodiments are provided by the following numbered embodiments:

1. A method for delivering a payload to a cell, the method comprising: providing a cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising: a cell-surface-targeting domain (CSTD) that binds to one or more targets on a surface of a cell, wherein when the CSTD is bound to the target on the surface of the cell, it is internalized into the cell; a random copolymer conjugated to the CSTD, the random copolymer comprising multiple polymer arms extending from a branched initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer, wherein the first and second monomers are distributed randomly throughout the copolymer, and wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone; and a payload linked to the second monomer, wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio is between 1-400; and administering the CSTDPPC to a cell, thereby binding the CSTDPPC to the cell, wherein the binding results in internalization thereby internalizing the payload, and wherein the internalized payload is released from the polymer. la. A method for delivering a payload to a cell, comprising: providing a cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising: a cell-surface-targeting domain (CSTD) configured for binding to one or more targets on a surface of a cell; a copolymer conjugated to the CSTD, the copolymer comprising one or more polymer arms extending from an initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer; and one or more payloads linked to the copolymer and/or the CSTD, optionally wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400; and administering an effective amount of the CSTDPPC to a cell to thereby deliver the one or more payloads to the cell, optionally, wherein the CSTDPPC comprises a linker between the one or more payloads and the second monomer is configured to release the one or more payloads in the cell upon internalization following binding of the CSTD to the one or more targets on the surface of the cell. lb. The method of embodiment la, wherein the first and second monomers are distributed randomly throughout the copolymer. lc. The method of embodiment la or lb, wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone. ld. The method of any one of embodiments la-lc, wherein the one or more payloads are linked to the polymer via the second monomer. le. The method of any one of embodiments la- Id, wherein the CSTDPPC comprises a cleavable linker that covalently links the one or more payloads to the copolymer. lf. The method of any one of embodiments la-le, wherein the copolymer comprises multiple polymer arms extending from the initiator fragment. lg. The method of any one of embodiments 1 a- If, wherein initiator fragment is a branched initiator fragment.

2. The method of any one of the preceding embodiments, wherein the cell is within a subject.

3. The method of any one of the preceding embodiments, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the payload or the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment that conjugates with the cell-surface-targeting domain (200);

F is a radical scavenger, such that the combination of I-I’ is an initiator, I¹, for the polymerization of the copolymer of Formula I; subscripts x and y¹ are each independently an integer of from 1 to 1000; subscript z is an integer of from 1 to 10; subscript s is an integer of from 1 to 100; and subscript n is an integer of from 1 to 20.

3a. The method of any one of embodiments 1-3, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the CSTD or the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment, optionally wherein I conjugates with the cell-surface- targeting domain;

I’ is a radical scavenger, such that the combination of I-I’ is an initiator, I¹, for the polymerization of the copolymer of Formula I; subscripts x and y¹ are each independently an integer of from 1 to 1000; subscript z is an integer of from 1 to 10; subscript s is an integer of from 1 to 100; and subscript n is an integer of from 1 to 20.

4. The method of any one of the preceding embodiments, wherein the copolymer is represented by Formula II: (Formula II), wherein each of R¹ and R² is H or C1-C4 alkyl, and PC is phosphorylcholine.

5. A cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising: a cell-surface-targeting domain (CSTD) that is internalized upon binding to a target on a cell surface; a random copolymer conjugated to the CSTD, wherein the random copolymer comprises multiple polymer arms extending from a branched initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer, wherein the first and second monomers are distributed randomly throughout the copolymer, and wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone; and a payload covalently linked to the second monomer, optionally wherein the payload-to-cell-surface-targeting domain (“PC STD”) ratio is between 1-400, and wherein upon binding to the target on the cell surface the CSTDPPC is internalized thereby internalizing the payload.

5a. A cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising: a cell-surface-targeting domain (CSTD) configured for binding to one or more targets on a cell surface; a copolymer conjugated to the CSTD, wherein the copolymer comprises multiple polymer arms extending from an initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer; and one or more payloads linked to the copolymer and/or the CSTD, optionally wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400, optionally wherein upon binding to the target on the cell surface the CSTDPPC is internalized thereby internalizing the payload.

5b. The CSTDPPC of embodiment 5a, wherein the first and second monomers are distributed randomly throughout the copolymer.

5c. The CSTDPPC of embodiment 5a or 5b, wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone.

5d. The CSTDPPC of any one of embodiments 5a-5c, wherein the one or more payloads are linked to the polymer via the second monomer.

5e. The CSTDPPC of any one of embodiments 5a-5d, wherein the CSTDPPC comprises a cleavable linker that covalently links the one or more payloads to the copolymer.

5f. The CSTDPPC of any one of embodiments 5a-5e, wherein the copolymer comprises multiple polymer arms extending from the initiator fragment.

5g. The CSTDPPC of any one of embodiments 5a-5f, wherein initiator fragment is a branched initiator fragment.

6. The CSTDPPC of any one of embodiments 5-5g, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone; M² links to the payload or the one or more payloads;

ZW is a zwitterionic moiety;

6a. The CSTDPPC of any one of embodiments 5-5g, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the CSTD or the payload or the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment;

7. The CSTDPPC of any one of embodiments 5-6a, wherein the copolymer is further represented by Formula II: (Formula II), wherein each of R¹ and R² is H or C1-C4 alkyl, and PC is phosphorylcholine.

8. The method of any one of embodiments 1-4 or the CSTDPPC of any one of embodiments 5-7, wherein the cell-surface-targeting-domain (CSTD) is a protein selected from the group consisting of antibodies, single-chain Fvs, Fabs, single domain antibodies, centyrins, nanofitins, affibodies, DARPins, affilins, anticalins, Kunitz, avimers, monobodies, peptides, cyclic peptides, trap domains, trap-domain-antibody fusions, knottins, and native and engineered proteins that to bind a target or targets on the surface of a cell.

9. The method or CSTDPPC of embodiment 8, wherein the cell-surface-targeting- domain is an antibody or an antigen-binding fragment thereof.

10. The method or CSTDPPC of embodiment 9, wherein the antibody is an anti- VEGFR2-antib ody .

11. The method or CSTDPPC of embodiment 9, wherein the antibody is an anti- TfRl -antibody.

12. The method or CSTDPPC of embodiment 9, wherein the antibody is an anti- HER2-antibody

13. The method of any one of embodiments 1-4 or the CSTDPPC of any one of embodiments 5-7, wherein the CSTD is selected from the group consisting of aptamers, sugar moieties, macrocycles, and lipids.

14. The method or CSTDPPC of embodiment 13, wherein the CSTD is an aptamer.

15. The method or CSTDPPC of embodiment 14, wherein the aptamer targets TfRl.

16. The method or CSTDPPC of any one of embodiments 8-15, wherein targeting is tissue specific.

17. The method of any one of embodiments 1-4 or the CSTDPPC of any one of embodiments 5-7, wherein the payload is selected from the group consisting of oligonucleotides, small molecules, proteins, peptides, aptamers, macrocycles, cyclic peptides, tRNAs, dye/label, or combinations thereof. 18. The method or CSTDPPC of embodiment 17, wherein the payload is an oligonucleotide.

19. The method or CSTDPPC of embodiment 18, wherein the oligonucleotide is a PCSK9 siRNA.

20. The method or CSTDPPC of embodiment 18, wherein the oligonucleotide is a PCSK9 antisense oligonucleotide.

21. The method or CSTDPPC of embodiment 17, wherein the payload is a small molecule.

22. The method or CSTDPPC of embodiment 21, wherein the small molecule is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

23. A cell-surface-targeting-domain-polymer-fluorescent dye (CSTDPFDC) conjugate comprising: a CSTD comprising an anti-VEGFR2 antibody; a polymer, wherein the polymer is a homopolymer, and wherein the polymer is conjugated to the anti-VEGFR2 antibody; and a fluorescent dye, wherein the fluorescent dye is covalently linked to the antibody.

23 a. A cell-surface-targeting-domain-polymer-fluorescent dye (CSTDPFDC) conjugate comprising: a CSTD comprising an antibody; a polymer conjugated to the antibody; and a fluorescent dye, wherein the fluorescent dye is covalently linked to the antibody and/or the polymer.

23b. The CSTDPFDC of embodiment 23a, wherein the polymer is a homopolymer.

23c. The CSTDPFDC of embodiment 23a, wherein the polymer is a copolymer.

23d. The CSTDPFDC of any one of embodiments 23a-23c, wherein the fluorescent dye is covalently linked to the antibody.

23e. The CSTDPFDC of any one of embodiments 23a-23c, wherein the fluorescent dye is covalently linked to the polymer.

24. The method or the CSTDPPC or the CSTDPFDC according to any one of the preceding embodiments, wherein the polymer or copolymer has a molecular weight between about 10,000 and about 1 ,750,000 Da as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC-MALS”).

25. The method or the CSTDPPC or the CSTDPFDC of embodiment 24, wherein the polymer or copolymer has a molecular weight of about 10,000 Da, or about 25,000 Da, or about 38,000 Da.

26. The method or the CSTDPPC or the CSTDPFDC of embodiment 24, wherein the polymer or copolymer has a molecular weight of about 50,000 Da.

27. The method or the CSTDPPC or the CSTDPFDC of embodiment 24, wherein the polymer or copolymer has a molecular weight of about 100,000 Da.

28. The method or the CSTDPPC or the CSTDPFDC of embodiment 24, wherein the polymer or copolymer has a molecular weight of about 150,000 Da, or about 180,000 Da, or about 290,000 Da.

29. The method or the CSTDPPC or the CSTDPFDC of embodiment 24 wherein the polymer or copolymer has a molecular weight of about 250,000 Da.

30. The method or the CSTDPPC or the CSTDPFDC of embodiment 24 wherein the molecular weight of the polymer is 300,000 Da or greater.

31. The method or the CSTDPPC or the CSTDPFDC of any one of the preceding embodiments, wherein the polymer or copolymer has a molecular weight between about 500,000 and about 1,000,000 Da.

32. The method or the CSTDPPC or the CSTDPFDC of any one of the preceding embodiments, wherein the polymer or copolymer has a molecular weight between about 750,000 to about 850,000 Da.

33. The method or the CSTDPPC of any one of the preceding embodiments, wherein the PCSTD ratio is between 5-400.

34. The method or the CSTDPPC of any one of the preceding embodiments, wherein the PCSTD ratio is between 10-400.

35. The method or the CSTDPPC of any one of the preceding embodiments, wherein the PCSTD ratio is between 20-400.

[0151] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0152] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0153] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0154] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXAMPLES

Example 1. The impact of conjugation on antigen binding.

[0155] This non-limiting example shows the impact of biopolymer conjugation on the binding of an antibody biopolymer conjugate (e.g., ABC) to surface antigen.

[0156] The binding of an anti-VEGF antibody that has the sequence shown in FIG. 9 and its biopolymer conjugate (e.g., ABC) to VEGF coating on a streptavidin plate (FIG. 2A) and a MaxiSorp plate (FIG. 2B) was tested. For ELISA with MaxiSorp plate (Cat. # 442404, Thermo Fisher Scientific, Inc), the plate was coated with 50 pL of 2 pg/mL human biotinylated VEGF 165 (R&D). For ELISA with streptavidin plate (Cat. # 15120, Thermo Fisher Scientific, Inc), 50 pL of human biotinylated VEGF 165 at 0.5 pg/mL was used. The procedures are the same for both assays. The antigens were coated on the plates at 4° C overnight. After three washes with lx PBST, the plates were blocked with 2% BSA in PBS for I~2 hours. Anti- VEGF antibody and its biopolymer conjugate (e.g., ABC) were 3-fold titrated starting from 30 nM to 0.04 nM. After the plates were washed three times with lx PBST, 50 pL of the titrated antibodies was added to the blocked wells and incubated at room temperature for 2 hours. After three washes, 50 pL of goat anti-human-HRP detection antibody (Cat. # ab97225, Abeam) at 1 : 10000 dilution was added for a 45 minutes incubation. After three washes, the binding signal was developed with SureBlue™ TMB substrate (Cat. # 52- 00-02, KPL). The absorbance at 450 nm was measured on an iD3 plate reader (Molecular Devices) after the reaction was stopped. The data were plotted as the average of replicates against the log of antibody concentrations using GraphPad Prism and fitted with a non-linear regression curve. The result shows the binding signal on streptavidin plate is overall higher than the signal on the MaxiSorp plate (FIG. 2A and FIG. 2B), even though VEGF coating concentration for streptavidin plate (0.5 pg/mL) is lower than for MaxiSorp plate (2 pg/mL). Binding of the antibody biopolymer conjugate (e.g., ABC) to VEGF on the surface in both assays was compromised, which is likely due to the steric hindrance between the polymer and the plate surface.

[0157] The antigen in-solution binding was evaluated by competition ELISA (FIG. 2C). A Nunc 96-well MaxiSorp plate was coated with 50 pL of human VEGFR1 at 1 pg/mL (Cat. # MAB321, R&D) at 4 °C overnight. The plate was blocked with 2% BSA in PBS for I~2 hours, followed by washing three times with lx PBST. The antibodies were 3-fold titrated and L I mixed with human biotinylated VEGF165, which yielded the final concentration of biotinylated VEGF 165 at 100 pM and titrated antibodies from 30 nM to 0.04 nM. The mixture was incubated for 30 minutes at room temperature and then added to the blocked wells. After 1.5 hours incubation at room temperature, the plate was washed three times with lx PBST. The captured biotinylated VEGF165 was detected with Pierce™ high sensitivity streptavidin- HRP (Cat. # 21130, Thermo Fisher Scientific, Inc) at 1:5000 dilution. The binding signal was developed with SureBlue™ TMB substrate. After stopping the reaction, absorbance at 450 nm was measured on an iD3 plate reader. The competition curve was plotted as the average reading signal of replicates against the log of antibody concentrations in FIG.2C. In the in-solution competition ELISA, anti-VEGF antibody and its biopolymer conjugate (e.g., ABC) show comparable potency on inhibition of VEGF binding to the receptor.

[0158] Thus the above shows that, in solution, the biopolymer-conjugated anti- VEGF antibody (e.g., ABC) exhibits similar binding potency to the unconjugated antibody.

Example 2, Anti-VEGFR2 antibody production and characterization.

[0159] This non-limiting example shows production and characterization of binding affinity for an anti-VEGFR2 antibody.

[0160] An anti-VEGFR2 antibody, known to be internalized upon VEGFR2 binding, was produced in Expi293 cells by transient transfection and purified by MabSelect PrismA column (Cytiva). The potency of the purified antibody was characterized by competition ELISA (FIG. 3). Briefly, a Nunc 96-well MaxiSorp plate was coated with 50 pL of 2 pg/mL human VEGFR2/KDR protein (Cat. # 357-KD-050/CF, R&D) at 4 °C overnight. The plate was blocked with 2% BSA in PBS for I~2 hours, followed by three washes with lx PBST. Anti-VEGFR2 antibody in 3-fold titrations were mixed with biotinylated VEGF165, which yielded the final concentration of biotinylated VEGF at 100 pM and anti-VEGFR2 from 100 nM to 0.14 nM. 50 pL of the mixture was added to the blocked wells. After 1 hour incubation at room temperature, the plate was washed three times with lx PBST. The captured biotinylated VEGF 165 was detected with Pierce™ high sensitivity streptavidin-HRP at 1 : 5000 dilution for 45 minutes incubation. Data collection and analysis were performed according to described in example 1. Purified anti-VEGFR2 proteins show dose dependent competition on VEGF binding to VEGFR2 with ICso=5.96 nM in tested assay condition (FIG. 3). [0161] Thus, the above shows that the purified anti-VEGFR2 antibody shows potent binding affinity for VEGFR2.

Example 3, Conjugation of anti-VEGFR2 antibody with a biopolymer.

[0162] This non-limiting example shows production of an anti-VEGFR2 antibody biopolymer conjugate (e.g., anti-VEGFR2 ABC).

[0163] The conjugation process involved two steps: an initial reduction reaction to deprotect cysteine thiol side chains (decapping), followed by a conjugation to the biopolymer OG1802 (FIG. 26) via maleimide-cysteine chemistry. The decapping was done by reducing antibodies with 30x molar excess of Tris (2- carboxyethyl) phosphine hydrochloride (TCEP) for 1 hour at room temperature followed by buffer exchange into 20 mM Tris-HCl pH 7.5, 50 mM NaCl to remove the cysteine cap and TCEP. Then, the reduced antibodies were oxidized with 15x molar excess of dehydro-ascorbic acid (dHAA) for 1 hour at room temperature followed by buffer exchange into 20 mM Tris pH 7.5, 50 mM NaCl to remove the oxidizing reagent. The conjugation process was done by mixing antibodies at final concentration of 2.0 mg/mL (or 14 pM) with 5x molar excess of biopolymer (750 kDa) in 20 mM Tris pH 8.5, 50 mM NaCl, 2 mM EDTA. The conjugation process took place at 2-8 °C for a period of 2 to 3 days. Conjugated antibodies were purified from unconjugated material by cation exchange chromatography (CEX) with Poros XS 1 mL column (Thermo Fisher Scientific, Inc) using a step gradient of increasing salt concentrations (50-900 mM NaCl) in buffer containing 20 mM sodium phosphate pH 6.0. The fractions containing the biopolymer conjugated antibodies were pooled and buffer exchanged into lx PBS, pH7.4.

[0164] Thus the above shows that the purified anti-VEGFR2 antibody can be conjugated with the biopolymer.

Example 4, pHAb dye labeling.

[0165] This non-limiting example shows labeling of an antibody and an antibody biopolymer conjugate (e.g., ABC) with a pH sensitive fluorescence dye.

[0166] pHAb dye (Promega) is a pH sensitive fluorescence dye with excitation (Ex) maxima at 532 nm and emission (Em) maxima at 560 nm. The dye with low-pH-dependent fluorescence is designed for antibody internalization screening. The antibodies and biopolymer conjugates (e.g., ABCs) were labeled with pHAb Amine Reactive Dye (Cat. # G9841 , Promega) according to the manufacturer’s protocol. Briefly, the antibodies or biopolymer conjugates (e.g., ABCs) were mixed with 20x molar excess of the dye in amine conjugation buffer (10 mM sodium bicarbonate buffer, pH 8.5). The reactions were incubated at room temperature for 1 hour. The unreacted dye was removed by dialysis against lx PBS, pH7.4. The concentration of dye-labeled antibodies and dye to antibody ratio (DAR) were calculated as recommended by the manufacturer. The average DAR value for the labeled molecules is ~10.

[0167] To assess the pH response, labeled antibodies or biopolymer conjugates (e.g., ABCs) were diluted to 15 nM with the buffers at different pH conditions. The fluorescence was measured at Ex/Em: 532 nm/560 nm on Tecan Spark plate reader. The result shows the fluorescence of labeled molecules is low-pH-dependent and the signal is weak at neutral pH (FIG. 4).

[0168] Thus, the above shows that the anti-VEGFR antibody, its biopolymer conjugate and a control antibody can be labeled with a pH sensitive fluorescence dye and that they emit a fluorescence signal at low pH.

Example 5, Potency evaluation of pHAb labeled antibodies.

[0169] This non-limiting example shows the impact of biopolymer conjugation and pH sensitive fluorescence dye labeling on anti-VEGFR2 potency in inhibiting VEGF-induced VEGFR2/KDR signaling and in an ELISA (in solution binding) assay.

[0170] VEGF cell reporter assay VEGF bioassay kit (Cat. # G7940, Promega) was used to evaluate the potency of conjugated and labeled anti-VEGFR2 on inhibiting VEGF- induced VEGFR2/KDR signaling in KDR/NFAT-293 cells The assay was performed according to the manufacturer’s instruction. KDR/NFAT-HEK293 cells were quickly thawed and resuspended in the assay media (10% FBS in DMEM Medium). 25 pL of diluted human VEGF165 (Cat. # 293-VE-010/CF, R&D) was preincubated with 25 pL of 8-point, 2.5-fold serial diluted antibodies in the assay plate at 37 °C for 30 minutes. Then, the cells were seeded at the density of 40,000 cells per 25 pL per well on assay plate, which yielded the final concentration of VEGF 165 at 0.5 nM and antibody titrations from 75 nM to O.l nM. The assay plate was cultured at 37 °C in 5% CO2 humidified incubator. After 6 hours incubation, the assay plate was equilibrated to room temperature for 15 minutes. 75 pL of Bio-Gio Reagent mixture (Promega) was added to each well and incubated at room temperature for 5 minutes. The luminescence was measured by iD3 plate reader. Relative luminescence units (RLUs) were plotted as the average of replicates with standard error against the log of the concentrations of antibodies and biopolymer conjugates (e.g., ABCs) using GraphPad Prism. The plots were fitted with a non-linear regression curve (FIG.5A). The result shows labeling slightly decreases the potency on VEGFR2/KDR signaling inhibition. The conjugation shows an increased impact on the surface receptor binding, likely due to the steric hindrance of the attached polymer.

[0171] In-solution binding ELISA An optimized ELISA format with antibody and VEGFR2 binding in solution was used to evaluate the direct impact of conjugation and labeling on the potency. A Nunc 96-well MaxiSorp plate was coated with 50 pL of 1 pg/mL human VEGF165 at 4 °C overnight. The plate was blocked with 2% BSA in PBS for 1~2 hours, followed by washing the plate three times with lx PBST. Human VEGFR2/KDR protein at final concentration of 100 pM was preincubated with 3 -fold serial diluted antibodies or biopolymer conjugates (e.g., ABCs) at final concentration starting from 30 nM to 0.04 nM for 1 hour. 50 pL of the mixture was added to the blocked wells. After 1 hour incubation at room temperature, the plate was washed three times with lx PBST. The His-tag on captured VEGFR2/KDR protein was detected by anti-His-HRP (Cat. # A190-114P, Thermo Fisher Scientific, Inc) at 1 :2000 dilution. The signal developing and data analysis steps were performed as described in example 1. The competition curves were presented in FIG. 5B. Both conjugation and labeling show minor impact on the potency of anti-VEGFR2 (Table 3). The labeled negative control antibody (NC mab) shows minimal background.

Table 3, IC50 value by in-solution binding ELISA. * Not applicable

[0172] Thus, the above shows that conjugation of the biopolymer and the pH- sensitive dye to the anti-VEGFR2 antibody minimally decreases its potency in VEGFR2/KDR signaling and binding to VEGFR2 in solution.

Example 6, Antibody internalization assay.

[0173] This non-limiting example shows the internalization of antibody biopolymer conjugate .

[0174] KDR/NFAT-HEK293 cells were used to study the internalization of pHAb dye labeled anti-VEGFR2 and its biopolymer conjugate (e.g., ABC) (FIG. 6). An antibody that does not bind to targets on the cell surface was labeled and used as a negative control (NC mab). KDR/NFAT-HEK293_cells were quickly thawed and resuspended in the assay media (10% FBS, 90% DMEM Medium). The cell density was adjusted to 8 xlO⁵ cells/mL. Then, 50 pL of KDR/NFAT-HEK293 with 40,000 cells were seeded to wells on assay plate. 25 pL of the antibody serial dilutions were added to the plated cells, which yielded the final concentration of titrations from 75 nM to 0.1 nM. The assay plate was cultured at 37 °C in 5% CO2 humidified incubator for 20-24 hours. After removing the reaction from the assay plate, the assay buffer was added to each well for imaging the internalization signal with Nikon eclipse Ti2 microscope. After imaging, the assay buffer was replaced by PBS and the fluorescence was read on Tecan Spark plate reader at Ex/Em: 532 nm/560 nm. The specific and concentration dependent fluorescence signal could be detected for the cells treated with either anti-VEGFR2 or its biopolymer conjugate (e.g., ABC) (FIG. 7). As shown in FIG. 7, no concentration dependent fluorescence signal could be detected for the controls without cells (empty symbols: anti-VEGFR2-pHAb, anti-VEGFR2-ABC pHAb, NC mab-pHAb, buffer), or the control with only cells (filled upside-down triangle).

[0175] The fluorescent images show the punctate structures inside the cells treated with 75 nM anti-VEGFR2 antibody or its biopolymer conjugate (e.g., ABC), which indicates visible internalized molecules. No signal was seen for the NC mab treated cells or cell only controls (FIG. 8). There is no significant difference in the distribution or intensity of the internalized fluorescence between anti-VEGFR2 antibody and its biopolymer conjugate (e.g., ABC) under the tested conditions. [0176] Thus, the results show that the anti-VEGFR-antibody conjugated to both the biopolymer and the pH-sensitive dye is internalized into the internal cellular space.

Example 7: Internalization of antibody biopolymer conjugate via receptor-mediated mechanism

[0177] Purpose: Antibody-drug conjugates (ADCs) are a class of therapeutics that combine the target specificity of an antibody with a linked toxin. Conventional antibody drug conjugation methods have limited drug-to-antibody ratio (DAR), a critical attribute for ADC efficacy. In some embodiments of the antibody biopolymer conjugate (e.g., ABC) platform of the present disclosure, an antibody is conjugated to a large branched hydrophilic phosphorylcholine biopolymer. The ability to generate random copolymers allows high loading of small molecules to this attached biopolymer and may overcome DAR limitations without perturbing antibody function. Here, it was evaluated if the antibody biopolymer conjugates (e.g., ABCs) can be internalized upon binding to cell-surface receptors using a fluorescence-based internalization assay, providing a proof of concept for using antibody biopolymer conjugates (e.g., ABCs) for target-specific intracellular deliveries.

[0178] Methods: A known internalizing anti-VEGFR2 antibody was conjugated to an 800 kDa biopolymer. The antibody and its conjugate were labeled with pHAb amine reactive dye, and their potency was evaluated by ELISA and VEGF cell reporter assays. For the antibody internalization assay, VEGFR2 expressing cells were treated with pHAb labeled molecules. The internalization signal was monitored at Ex/Em: 532nm/560nm by Tecan plate reader and images were taken under RFP channel with a Nikon microscope.

[0179] Results: Dye labeling had minor impact on the potency of anti-VEGFR2 and its conjugate. The latter showed partially compromised binding to VEGFR2 on cell surface likely due to the steric hindrance of the attached polymer. Fluorescence measurements showed emission intensity of pHAb labeled molecules is pH dependent. In the internalization assay, a specific and concentration dependent fluorescence signal was detected for the cells treated with either anti-VEGFR2 or its conjugate. No significant difference was observed in the distribution or intensity of the internalized fluorescence between the anti-VEGFR2 antibody and its conjugate. [0180] Conclusions: The internalization of anti-VEGFR2 antibody biopolymer conjugate demonstrates the potential for intracellular drug delivery using the cell-surface- targeting-domain-polymer-payload conjugate (CSTDPPC) (e.g., ABC Platform™). This platform, combined with diverse drug modalities such as oligonucleotides, small molecules and peptides, overcomes current DAR limitations and provides a broad range of options for drug loading and intracellular delivery.

Example 8: Administer a molecule to a cell that is detectable.

[0181] Cell-surface-targeting-domain-polymer-payload conjugates (CSTDPPC) 100 FIG.l with fluorescent molecule (payloads, 400 FIG. 1) will be administered to cells, and cells will be visualized for a fluorescent signal. The payload-to-cell-surface-targeting domain (“PCSTD”) ratios (“PCSTD ratios”) will be in the range of 1-400. In some embodiments, the PCSTD ratio will be more than 1-400, for example 5, 10, or 20 or more to 400.

[0182] These results will show that a detectable payload-to-cell-surface-targeting domain (“PCSTD”) ratios can be achieved for the cell-surface-targeting-domain-polymer- payload conjugates (CSTDPPCs) 100 FIG. 1 with fluorescent molecule payloads 400, and that the CSTDPPCs can deliver detectable payloads 400 to the internal spaces of cells.

Example 9: Administer a molecule that has a biological effect on a cell.

[0183] Payload molecules 400 FIG. 1 will be delivered to cells via a cell-surface- targeting-domain-polymer-payload conjugate (CSTDPPC) 100 FIG. 1. The payload-to-cell- surface-targeting domain (“PCSTD”) ratios will be in the range of 1-400. In some embodiments, the PCSTD ratio is more than 1-400, for example 5, 10, or 20 or more to 400. The CSTDPPC will be administered until a biological or therapeutic response is observed.

[0184] These results will show that a therapeutically effective payload-to-cell- surface-targeting domain (“PCSTD”) ratios can be achieved for the cell-surface-targeting- domain-polymer-payload conjugates (CSTDPPCs) 100 FIG. 1 and that the CSTDPPCs can deliver therapeutically effective payloads 400 to the internal spaces of cells. Example 10: Generation and Characterization of Antibody Biopolymer Conjugate Oligonucleotides (e.g., ABCOs)

[0185] This non-limiting example shows generation and characterization of antibody biopolymer conjugate oligonucleotides (e.g., ABCOs).

[0186] PCSK9, a protein primarily produced by the liver, binds to LDL receptors on liver cells and promotes the degradation of LDLR, thus reducing the liver's ability to remove LDL from the bloodstream. Here an antibody biopolymer conjugate oligonucleotide (e.g., ABCO) molecule was designed to deliver PCSK9 siRNA or antisense oligonucleotide (ASO) into hepatic cells. Both siRNA and ASO contained bicyclononyne (BCN)-PEG4-Val-Cit-PAB linker where valine-citrulline (“Val-Cit”) protease-labile component was included to be cleaved by Cathepsin B in the lysosome. Anti-TfRl antibody was used to target the cell surface domain of the transferrin receptor 1 (TfRl), which is abundantly expressed in hepatocytes, such as HepG2 cells.

[0187] The conjugation process of anti-TfRl antibody biopolymer conjugate (e.g., ABC) was performed as described in Example 3. Briefly, the decapped and refolded antibody at a concentration of between 15 and 30 pM was mixed with 3x to 5x molar access of activated copolymers (Table 4) and the cysteine-maleimide reaction was allowed to proceed for 24 to 48 hours. The conjugation process was monitored by SDS-PAGE, and after completion, the antibody biopolymer conjugate (e.g., ABC) was purified using Protein-A affinity chromatography with Mab Select Sure resin using a step gradient of decreasing pH with buffers containing 50 mM citrate salts. After purification, antibody biopolymer conjugate (e.g., ABC) was solvent-exchanged into lx PBS, pH 7.4 and used in the next stage of payload clicking. Nucleic acid-based payload was clicked onto the anti-TfRl antibody biopolymer conjugate (e.g., ABC) intermediate. The anti-TfRl antibody biopolymer conjugate (e.g., ABC) was diluted to achieve a dissolved azide (-N3) concentration around 400 pM in acetate buffer pH 5.5. The headspace above the solution was sparged with dry nitrogen gas to remove all dissolved oxygen. The required amount of BCN-terminated ASO (Bio-Synthesis Inc.) or siRNA (Bio-Synthesis Inc) solution in water was added to the antibody biopolymer conjugate (e.g., ABC) such that molar excess of ASO or siRNA is achieved relative to the targeted number of azides. The reaction was allowed to proceed for 48 to 60 hours after which the mixture was purified using size exclusion chromatography. The molecular weights and DARs were determined by conjugation analysis on SEC-MALS (Table 5 and FIG. 10).

[0188] Nucleic acid payload release was tested using human Cathepsin B enzyme. For a typical procedure, 10 mM solution of human Cathepsin B enzyme (ACROBiosystems) in a formulation buffer was activated using 10 mM dithiothreitol (DTT) with 2 mM EDTA solution for 15 min at room temperature according to manufacturer’s instructions. Copolymernucleic acid conjugate (BCO1) was solvent-exchanged into 50 mM acetate buffer, pH 5.0 and 0. lx molar access of Cathepsin B was added to the solution. Reaction progress was monitored using size exclusion chromatography connected to a UV detector over the course of two hours by repeatedly injecting small aliquots of the reaction onto the SEC column. The majority of siRNA molecules were released from copolymer by Cathepsin B in 20 minutes (FIG. 11). Thus, reaction kinetics confirms efficient enzymatic cleavage of RNA-copolymer conjugate in the presence of human Cathepsin B at pH 5 (FIG. 11).

Table 4, List of molecules used in examples.

*DARs were measured as described in Table 5 and Table 10.

Table 5, SEC-MALS conjugates characterization data

[0189] Thus, the above shows that antibodies can be conjugated with copolymers and oligonucleotides, and that enzymatic release of the oligonucleotides is efficient.

Example 11 : Evaluation of anti-TfRl -antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oligonucleotides (e.g., ABCOs) binding to TfRl

[0190] This non-limiting example shows binding kinetics of anti-TfRl ABCs and ABCOs to free and surface-bound TfRl protein.

[0191] Binding kinetics of an antibody biopolymer conjugate oligonucleotide (e.g., ABCO) molecules to free TfRl protein were tested at 37 °C using a Biacore T200. Anti-TfRl antibody, ABC1, ABC2 and ABCO1 (see Table 4) were captured on a series S sensor Protein A chip (Cytiva) at flow rate of 10 pL/min for 20 seconds. Recombinant human TfRl-His protein (huTfRl-His, Cat. #CD1-H5243, ACROBiosystems) was titrated down by 3-fold serial dilution in HBS-EP+ buffer, ranging from 3.3 nM to 270 nM. The titrations were sequentially flowed over anti-TfRl antibody or its conjugates captured surface for 60 seconds at a flow rate of 30 pL/min, followed by 10-minute dissociation phase. The sensor chip surface was regenerated with 60-second injection of 10 mM glycine (pH 1.7) at a flow rate of 50 pL/min. All sensorgrams were double reference-subtracted and fitted using a 1 : 1 Langmuir binding model (FIG. 12A-12D). The binding kinetic results, shown in Table 6, indicate that conjugation of different sizes of copolymer and/or siRNA to anti-TfRl antibody did not affect its binding to the free TfRl protein. Table 6, Binding kinetic of anti-TfRl antibody and its conjugates for free TfRl protein.

*The kinetic constants cannot be uniquely determined.

[0192] To test the binding to surface bound TfR.1 protein, huTfRl-His protein was immobilized on series S sensor CM5 chips (Cytiva) by the amine coupling. Briefly, the chip surfaces were activated with EDC and NHS. TfRl-His protein (20 pg/mL) in 10 mM acetate pH 5.5 was injected at 10 pL/min flow rate for 12 seconds to achieve -300-600 RU. Anti-TfRl antibody, ABC1, AOC1, ABCO1 and ABCO2 (see Table 4) were 3-fold serial titrated inHBS- EP+ buffer and injected at increasing concentrations, from 1.2 nM to 100 nM, at 30 pL/min for 60 seconds followed by a 600 seconds dissociation. The sensor chip surface was regenerated by injecting 1.33 M NaCl/IgG elution buffer (Cat. # 21004, Thermo Fisher Scientific) for 30 seconds at a flow rate of 50 pL/min.

[0193] All sensorgrams were double reference-subtracted and fitted using a 1 : 1 Langmuir binding model (FIG. 13 and FIG. 14). The kinetics results were shown in Table 7.

[0194] In the surface bound TfRl binding test, the association rate (ka) and Rmax (RU) of antibody biopolymer conjugate (e.g., ABC) or antibody biopolymer conjugate oligonucleotides (e.g., ABCOs) indicated binding at a reduced level compared to anti-TfRl antibody, likely due to the steric hindrance from its increased size. For antibody biopolymer conjugate oligonucleotide (e.g., ABCO) molecules, negative charge may have contributed to this effect. With decreasing the copolymer size, the binding signal and Rmax of ABCO3 (see Table 4) were improved (Table 7 and FIG. 14C).

Table 7, Binding kinetic of anti-TfRl antibody and its conjugates for surface bound TfRl protein.

[0195] Thus, the binding kinetic results indicate that different sizes of copolymer and/or siRNA to anti-TfRl antibody can be conjugated while retaining effective binding to the free TfRl protein.

Example 12: Internalization of anti-TfRl -antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oli onucleotide (e.g., ABCQl-siRNAs.

. This non-limiting example shows the internalization of an antibody biopolymer conjugate (e.g. ABC) and an antibody biopolymer conjugate oligonucleotide (e.g. ABCO)- siRNA.

[0196] To determine the receptor mediated cellular uptake, we tested the internalization of anti-TfRl antibody, ABC1 and ABCO1 (see Table 4) in HepG2 cells. All samples were labeled with pH sensitive fluorescence dye pHAb as described in Example 4.

[0197] HepG2 cells (Cat. # HB-8065, ATCC) were cultured in complete growth media with Eagle’s Minimum Essential Medium (EMEM) supplemented 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 pg/ml streptomycin at 37 °C in a 5% CO2 humidified incubator. In the internalization assay, black 96-well plates (Cat. # 3904, Corning) were preseeded with 2 xlO⁴ cells per well on the day before. The pHAb dye labeled reagents were 4- fold titrated from 100 nM to 6.25 nM in EMEM medium. The cells were washed with DPBS and incubated with 100 pL of titrated reagents for 4 hours and then 10% FBS was supplemented followed by additional 16-20 hours incubation at 37 °C in 5% CO2 humidified incubator. After the treatment, the assay plates were washed twice with DPBS before the fluorescence was measured on Tecan Spark plate reader at Ex/Em: 532 nm/560 nm.

[0198] The result shows that anti-TfRl ABC1 and ABCO1 can be internalized into HepG2 cells. No signal was detected for the negative control (NC) antibody treatment (FIG. 15). Example 13: vitro gene knockdown assay with anti-TfRl -antibody biopolymer conjugate oligonucleotide (e.g., ABCOj-siRNAs

[0199] This non-limiting example evaluates the potency of anti-TfRl -antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-siRNA. This example relates to Example 10.

[0200] In vitro PCSK9 gene knockdown potency of anti-TfRl -antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-siRNA was assessed in HepG2 cells. The cells were seeded at a density of 1 xlO⁴ cells per well in a 96-well tissue culture plate for 18- 24 hours. After washing with DBPS, 100 pL of Opti-MEM media (Gibco) containing anti- TfRl ABC1, AOC1, ABCO1, ABCO2 or ABCO3 (see Table 4) were added to the cells and cultured for three days. The concentration of conjugated molecules was tested at 45 nM or across 25-fold serial titration range from 62.5 nM to 0.004 nM (ABCO3). The cells were then switched to complete growth media for an additional two days before being harvested in RNA shield solution (Cat. # R1100-50, Zymo).

[0201] siRNA and siRNA-copolymer were also evaluated by reverse transfection in HepG2 cells using Lipofectamine™ RNAiMAX (Cat. # 13778-075, Thermo Fisher Scientific). The siRNA samples were diluted into 200 pL of Opti-MEM medium and incubated with 2.5 pL of Lipofectamine RNAmaxi reagent for 10-20 mins on 12-well tissue culture plate. HepG2 cells at 1 xlO⁵ in 1 mL of complete growth medium without antibiotics were added to the wells, which gave a final siRNA concentration of 1 nM. The cells were cultured at 37 °C for three days before harvest.

[0202] The total RNA was extracted from the harvested cells using Quick-RNA Miniprep Kit (Cat. # R1054, Zymo) or Quick-RNA 96 Kit (Cat. # R1052, Zymo) according to the manufacturer’s protocols. The extracted RNA was subsequently used as a template for cDNA synthesis, performed with High-Capacity cDNA Reverse Transcription Kit (Cat. # 4368814, Thermo Fisher Scientific) as described in the manufacturer’s instructions. The PCSK9 mRNA level was measured by quantitative Real-time PCR (qPCR). The multiplex qPCR reactions were carried out with TaqMan Fast Advanced Master Mix (Cat. # 4444557; Thermo Fisher Scientific), TaqMan Gene Expression Assays probe [PCSK9 (FAM/MGB probe, Hs00545399_ml) and endogenous control GAPDH (VIC™/MGB probe, Hs99999905_ml), Thermo Fisher Scientific], cDNA template and nuclease-free water according to the manufacturer’s instruction. The thermal cycling program was set as follows: an initial 20 s denaturation at 95°C, followed by 40 cycles of 1 s at 95°C and 20 s at 60°C on QuantStudio™ 5 Realtime PCR system (Thermo Fisher Scientific). The PCSK9 mRNA levels were quantified using the 2 ^{AAC I} method with normalization to endogenous control GAPDH mRNA level. The results were presented as a percentage expression of PCSK9, relative to untreated control cells.

[0203] In the transfection assay, copolymer conjugated siRNA shows comparable potency as free siRNA payload. The PCSK9 mRNA level was reduced by -80% with 1 nM siRNA (FIG. 16A). This indicates conjugation of siRNA onto the copolymer did not impact its activity. In the cell treatment assay, ABCO1 reduced PCKS9 mRNA levels (FIG. 16B). In the treatment with a serially titrated ABCO3, the inhibition level was up to 50% (FIG. 17A).

[0204] Thus, the PCSK9 siRNA used is highly potent, requiring a very low siRNA concentration (<lnM) on ABCO3 to effectively titrate down the knockdown signal (FIG.17B).

Example 14: Internalization and in vitro gene knockdown with anti-TfRl -antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oligonucleotide (e.g., ABCO)- ASOs.

[0205] This non-limiting example evaluates the internalization of anti-TfRl - antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oligonucleotide (e g., ABCO)-ASOs and their potency in in vitro PCSK9 gene knockdown. This example relates to Example 10.

[0206] A PCSK9 ASO was used to generate anti-TfRl -antibody biopolymer conjugate oligonucleotide (e.g., ABCO) molecules. The internalization and in vitro PCSK9 gene knockdown were tested in HepG2 cells as described in Example 13.

[0207] In the internalization assay, anti-TfRl -antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-ASOs with different DARs or different copolymer sizes were labeled with pHAb low pH dye and 5-fold titrated from 100 nM to 0.16 nM in Opti-MEM medium. 100 pL of each titration was added to pre-seeded HepG2 cells for 20-24 hours treatment at 37 °C. The fluorescence reading indicates ASO DAR and copolymer size did not show significant impact on the internalization of antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-ASOs after up to 24-hour incubation. The negative control antibody and antibody biopolymer conjugate (e.g., ABC) did not show any fluorescence signal (FIG. 18A and 18B).

[0208] In the in vitro gene knockdown assay, anti-TfRl -antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-ASOs with different DARs (ABCO5, ABCO7 or ABCO8 (see Table 4)) were administered to HepG2 cells in Opti-MEM medium at concentrations of 200 nM, 20 nM and 2 nM. After 3 days, the treatments were replaced with complete growth media for an additional two days. The qPCR analysis revealed that anti-TfRl antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-ASOs exhibited concentrationdependent knockdown effect, with higher DAR yielding greater potency (FIG. 19).

[0209] Thus, the above shows efficient internalization of anti-TfRl -antibody biopolymer conjugates (e.g., ABCs) and antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-ASOs with different DARs or different copolymer sizes and a concentrationdependent knockdown effect, with higher DAR yielding greater potency.

Example 15: Generation of anti-TfRl-Aptamer copolymer conjugates

[0210] This non-limiting example shows generation of anti-TfRl-Aptamer copolymer conjugates.

[0211] Aptamers are single-stranded DNA or RNA molecules that bind to protein targets by folding into a three-dimensional conformation like antibodies. Here a TfRl targeting aptamer with a thiol group at the 5’ end (synthesized by IDT DNA, Inc.) was used as a cell surface targeting domain and conjugated to copolymer.

[0212] The anti-TfRl aptamer was structurally folded in 1 x PBS, pH 7.4 with 2 mM MgCk by heating at 95°C for 5 min on PCR machine and gradually cooling to the room temperature. The aptamer was conjugated to copolymerl via maleimide-thiol chemistry. The thiol group on aptamer was decapped by incubating the aptamer with 3 Ox molar excess of TCEP for 1 hour at room temperature followed by buffer exchange into aptamer conjugation buffer containing 20 mM Tris-HCl pH 8.5, 50 mM NaCl, 2 mM MgCh. The conjugation process was done by mixing aptamer at final concentration of 20 pM with 3 x molar excess of activated copolymerl at room temperature for one day. ABC5 (see Table 4) was purified from unconjugated material by anion exchange chromatography (AEX) with HiTrap Capto Q column, 1 mb (Cat. # 11001302, Thermo Fisher Scientific) using an increased salt gradients (125-750 mM NaCl) in buffer containing 20 mM Tris pH 8.5 and 2 mM MgCL. The fractions containing ABC5 were pooled and stored into conjugation buffer. Conjugation analysis by SEC-MALS indicates ABC5 contains one aptamer per copolymer.

[0213] To click the anti-TfRl aptamer on the copolymer4 (38 kDa; see Table 4), DBCO-PEG12-maleimide linker (Cat. # BP-25730, BroadPharm) was conjugated to aptamers by incubating 50 pM decapped anti-TfRl aptamer with the 3x molar excess of DBCO-PEG12- Maleimide at room temperature for one day in aptamer conjugation buffer. Anti-TfRl aptamer- DBCO was loaded on the copolymer4 via DBCO-Azide click reaction, by incubating 3x molar excess of anti-TfRl aptamer with 20 pM copolymer4 at RT for one day. ABC6 product (see Table 4) was separated by size exclusion chromatography using Superdex 200 increase 10/30 GL in a running buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCb. The fractions containing anti-TfRl aptamer ABC6 were pooled and stored into conjugation buffer. The ratio of aptamer to copolymer in ABC6 was assessed by SEC-MALS conjugation analysis, which shows each copolymer was loaded with 2-3 aptamers.

Example 16: Binding of anti-TfRl aptamer-biopolymer conjugate (e.g., ABC) to surface bound TfRl protein

[0214] This non-limiting example evaluates the binding kinetics of of anti-TfRl aptamer-biopolymer conjugate (e.g., ABC) to surface bound TfRl protein.

[0215] Binding kinetics assay was performed at 37 °C using a Biacore T200. The human TfRl-Fc protein (huTfRl-Fc, Cat. # H5264, ACROBiosy stems) at 2 pg/mL was captured on a Protein A chip at flow rate of 10 pL/min for 30 seconds. Free anti-TfRl aptamer or aptamer on biopolymer conjugate (e.g., ABC) were 3-fold titrated down in HBS-EP+ buffer supplemented with 5 mM MgCh, range from 1000 nM to 12.4 nM (ABC5; see Table 4) or 500 nM to 6.2 nM (ABC6; see Table 4), and flowed over captured TfRl-Fc for 90 at a flow rate of 30 pL/min followed by 4-minute dissociation. Sensor chip surface was regenerated by 60-second injection of 10 mM Glycine, pH 1.7 at a flow rate of 50 pL/min. All sensorgrams were double reference-subtracted and fitted using a 1 :1 Langmuir binding model. Kinetics results show the association rate of ABC5 decreased by ~15 folds compared to free anti-TfRl aptamer (Table 8 and FIG. 20A and 20B). Conjugation of a large size copolymer to a small binding motif could introduce more steric hindrance and decrease the binding affinity. By clicking onto a smaller size copolymer, introducing spacers between copolymer and aptamer, also loading multiple copies of aptamer, ABC6 showed improved binding affinity compared to ABC5 (Table 9, FIG. 21A-21B)

Table 8, Binding kinetic of anti-TfR.1 aptamer and ABC5 for surface bound TfRl protein

Table 9, Binding kinetic of anti-TfRl aptamer and ABC6 for surface bound TfRl protein

[0216] Thus, the above shows that aptamer binding affinity may be improved by clicking onto smaller size copolymers, introducing spacers between copolymers and aptamers, and loading multiple copies of aptamers.

Example 17: Internalization of anti-TfRl -Aptamer biopolymer conjugates (e.g., ABCs) in HepG2 cells

[0217] This non-limiting example evaluates internalization of anti-TfRl -Aptamer biopolymer conjugates (e.g., ABCs). This examples relates to Example 8.

[0218] To test the internalization of anti-TfRl aptamer biopolymer conjugate (e.g., ABC), the low pH SiteClick™ sDIBO Alkyne-pHrodo deep Red dye (Cat: S10914, Thermo Fisher Scientific) was labeled on copolymer in the conjugation buffer at pH 7.5. 10-20x molar excess of dye was incubated with anti-TfRl -Aptamer biopolymer conjugates (e.g., ABCs) or copolymer controls at RT for one day. To label the free aptamer, an Azido-PEG3 -Mai eimide linker was conjugated to the aptamer followed by clicking with sDIBO pHrodo Deep Red dye. The free dye was removed by Vivaspin500-10 kDa (Cytiva). The dye of labeling (DOL) was confirmed at OD640 absorbance according to the manufacturer’s instructions. The dye-to- molecule ratio was 0.4 for anti-TfRl aptamer, 1.2 for ABC5, and 2.6 for ABC6 (see Table 4).

[0219] For the internalization assay, black 96-well plates were pre-seeded with 2 xlO⁴ cells per well for 18-24 hours. The labeled reagents were 3-fold titrated in Opti-MEM medium with 5 mM MgCh, concentration range from 500 nM to 2 nM. The cells were washed with DPBS and treated with 100 pL of labeled reagents. The assay plates were cultured at 37 °C in 5% CO2 humidified incubator for 20-24 hours. After removing the reactions, the assay plates were washed 3-5 times with DPBS and the fluorescence was measured on Tecan Spark plate reader at Ex/Em: 635 nm/670 nm. The data was plotted in GraphPad Prism after subtracting the background signal of copolymer controls. Compared to free anti-TfRl aptamer, ABC5 shows weak internalization signal (FIG. 22A). After optimizing the design, ABC6 had great improvement in internalization (FIG. 22B).

[0220] Thus, the above shows that the optimization steps taken in Example 16 may also improve internalization.

Example 18: Generation of anti-HER2 Antibody Biopolymer Conjugate Drugs (e.g., ABCDs) [0221] This non-limiting example shows generation of anti-HER2 Antibody Biopolymer Conjugate Drugs (e.g., ABCDs).

[0222] To give proof of the concept for antibody biopolymer conjugate drug (e.g., ABCD)-mediated small molecule drug delivery, anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) molecules (see Table 4) were generated by loading copolymer with tubulin inhibitor monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). The drugs employed are endo-BCN-Val-Cit-PAB-MMAE (Cat. # BP-28192, BroadPharm), endo-BCN- PEG4-Val-Cit-PAB-MMAE (Cat. # BP-24415, BroadPharm), and endo-BCN-PEG4-Val-Cit- PAB-MMAF (Cat. # BP-29649, BroadPharm). A valine-citrulline protease-labile component was included in the linker between drug and copolymer, which could be cleaved by Cathepsin B in the lysosome to release the toxin.

[0223] The process of antibody biopolymer conjugate drug (e.g., ABCD) generation involved two steps: an initial drug loading onto the copolymer followed by conjugating the copolymer with anti-HER2 antibody via maleimide-cysteine chemistry. Drug loading onto copolymer was done by mixing copolymer5 at a final concentration of 10 mg/mL with 3x molar excess of a drug functionalized with a BCN functional group in 20 mM acetate buffer, 70 mM NaCl, pH 5.0 for 72 hours followed by buffer exchange into 0.5 M pH 7.8 Na phosphate, 30 mM sodium dihydrogen phosphate, 470 mM disodium hydrogen phosphate to remove the free drug reagents.

[0224] Free copolymer and drug loaded copolymers were activated by mixing with lOx molar excess of Snap-on 3 -maleimidopropionic acid, NHS ester for 30 minutes at 25 °C in 0.5 M pH 7.8 Na phosphate, 30 mM sodium dihydrogen phosphate, 470 mM disodium hydrogen phosphate. Followed by buffer exchange into 20 mM Tris pH 8.5, 50 mM NaCl, 2 mM EDTA.

[0225] The conjugation reactions were prepared as described in Example 3. The antibodies were mixed with 3.5x molar excess of activated unloaded (or loaded) copolymer5 in 20 mM Tris pH 8.5, 50 mM NaCl, 2 mM EDTA. The conjugation process took place at 25 °C for a period of 24 hours. Once the reactions were complete, antibody biopolymer conjugate (e.g., ABC) (or antibody biopolymer conjugate drugs (e.g., ABCDs)) were purified from unconjugated material by Protein A chromatography with HiTrap MabSelect SuRe 1 mL column (Cytiva US LLC) using a step pH gradient. Step 1 is 50 mM citrate pH 6.2 to elute free copolymer. Step 2 is 30% 50 mM citrate pH 6.22 & 70% 50 mM citrate pH 3.5 to elute antibody biopolymer conjugate (e.g., ABC) (or antibody biopolymer conjugate drug (e.g., ABCD)). Step 3 is 50 mM citrate pH 3.5 to elute free antibodies. The fractions containing the antibody biopolymer conjugate (e.g., ABC) (or antibody biopolymer conjugate drug (e.g., ABCD)) were pooled and buffer exchanged into lx PBS, pH 7.4.

Example 19: Characterization of anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) concentrations and DARs

[0226] This non-limiting example characterizes anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) concentrations and DARs.

[0227] Antibody concentrations in anti-HER2 antibody biopolymer conjugate (e.g., ABC) or antibody biopolymer conjugate drugs (e.g., ABCDs) were determined by using SDS-PAGE. Anti-HER2 Antibody only at defined concentrations, antibody biopolymer conjugate (e.g., ABC), and antibody biopolymer conjugate drug (e.g., ABCD) solutions were reduced and heated at 90 °C for 5 minutes. Samples were then loaded onto a 24 well NuPAGE 4-12% Bis-Tris gel and run at 200V for 45 minutes. Gels were stained with SimplyBlue SafeStain for one hour followed by destaining overnight. The antibody only solutions prepared at defined concentrations were used to create a calibration curve that converts light chain band intensity to antibody loading. With the calibration curve, anti-HER2 antibody biopolymer conjugate (e.g., ABC), ABCD-MMAE, ABCD-PEG4-MMAE, and ABCD-PEG4-MMAF yielded antibody concentrations of 17.49, 0.78, 2.18, and 0.96 pM, respectively (Table 10).

Table 10. Concentration of antibody and drug in antibody biopolymer conjugate (e g., ABC) and antibody biopolymer conjugate drug (e.g., ABCD)

*Drug concentration and DAR measured by Cathepsin B release assay.

[0228] To determine DARs, anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) were incubated with Cathepsin B (ACROBiosystems) for 24 hours with a drug : Cathepsin B ratio of 100 : 1 in 25 mM sodium acetate pH 5.0. Anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) and Cathepsin B solutions were then injected onto a SEC-UV setup equipped with a Superdex 200 Increase 10/300 GL column (Cytiva) in PBS at 1 mL/min. The conjugate molecules elute first followed by the released drug molecules (FIG. 23A-23C). The drug only solutions prepared at defined concentrations were run on SEC-UV to create a calibration curve that converts raw integrated UV signal to drug concentration. With the calibration curve, anti-HER2 ABCD-MMAE, ABCD-PEG4-MMAE, and ABCD-PEG4- MMAF yielded drug concentrations of 106.1, 206.5, and 86.0 pM, respectively, which corresponds to a DAR of 137, 141, and 89, respectively (Table 10).

Example 20: Internalization of anti-HER2-and its biopolymer conjugate (e.g., ABC).

[0229] This non-limiting example evaluates internalization of an anti-HER2-and its ABC. [0230] Anti-HER2 antibody and its biopolymer conjugate (e.g., ABC) were labeled with pH sensitive fluorescence dye pHAb as described in Example 4. HER2 positive breast cancer cells SKBR3 (Cat. # HTB-30, ATCC) were used to study internalization of pHAb labeled anti-HER2 antibody and its biopolymer conjugate (e.g., ABC). HER2 negative breast cancer cells MCF7 (Cat. # HTB-22, ATCC) were used as a negative control. SKBR3 cells were grown in McCoy's media with 10% FBS and MCF7 cells were grown in EMEM with 10% FBS, 0.01 mg/ml human recombinant insulin. All media were supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin. Cells were seeded at a density of 2 xlO⁴ per well in black 96-well plates for 18-24 hours to attach. Anti-HER2-pHAb and anti-HER2-ABC- pHAb were 3-fold titrated from 100 nM to 0.4 nM in Opti-MEM medium. The cells were washed with DPBS and treated with 100 pL of labeled reagents. The assay plates were cultured at 37 °C in 5% CO2 humidified incubator for 20-24 hours. After removing the reactions, the assay plates were washed twice with DPBS, and the fluorescence was read on Tecan Spark plate reader at Ex/Em: 532 nm/560 nm.

[0231] The results show anti-HER2 antibody, and its biopolymer conjugate (e.g., ABC) were specifically internalized into SKBR3 (HER2+) cells with a concentration dependent signal (FIG. 24A). No internalization signal was detected in MCF7 (HER2-) cells (FIG. 24B)

Example 2 \ In vitro Cytotoxicity of anti-HER2-Antibody Biopolymer Conjugates Drug (e.g., ABCDs) in HER2 Overexpressing Cancer Cell Lines.

[0232] This non-limiting example evaluates cytotoxicity of anti-HER2-antibody biopolymer conjugate drugs (e.g., ABCDs) in HER2 overexpressing cancer cell lines. This example relates to Example 10.

[0233] The in vitro cytotoxicity of anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) molecules was evaluated in SKBR3 (HER2+) and MCF7 (HER2-) breast cancer cell lines by MTS cell proliferation assay. Briefly, the cells were seeded at a density of 2xl0⁴ per well in complete growth media in 96-well cell culture plates. After 24 hours, the old medium was removed, and fresh medium was added along with serially diluted ABCD1, ABCD2 and ABCD3 (see Table 4) at concentration of 10 nM, 1 nM, and 0.1 nM. Anti-HER2 antibody and its ABC were also included as controls. The cells were treated at 37 °C for 72 hours. Then each well was added with 20 pL of CellTiter 96 ® AQueous One Solution MTS Cell Proliferation Assay reagents (Promega). The 96-well plates were incubated for 2 hours before read at 490 nm. Cell viability was calculated as the percentage of internally untreated control cells. All three anti-HER2 antibody biopolymer conjugate drug (e.g., ABCD) molecules exhibited high cytotoxicity only in the HER2 overexpressing cell line SKBR3. Under all three testing concentrations of anti-HER2 antibody biopolymer conjugate drugs (e.g., ABCDs), the growth of SKBR3 cells was inhibited by 60-70% (FIG. 25A). No significant toxicity was observed in the HER2-negative cell line MCF7 for either anti-HER2 antibody biopolymer conjugate drugs (e.g., ABCDs) or controls (FIG. 25B).

[0234] Thus, anti-HER2-antibody biopolymer conjugate drugs (e.g., ABCDs) exhibited cytotoxicity only in HER2 overexpressing cancer cell lines.

Example 22: Delivery of Therapeutic Oligonucleotides by Antibody Biopolymer Conjugates

[0235] Purpose: Oligonucleotide-based therapeutics are promising modalities for the treatment of ocular diseases, enabling precise silencing of pathogenic genes. Antibody Oligonucleotide Conjugates (AOCs) are designed to enhance the intracellular delivery of oligonucleotides to the target cells. However, limited Drug-to-Antibody Ratio (DAR) in conventional AOCs poses significant challenges in optimizing therapeutic efficacy. Antibody Biopolymer Conjugate Drug (e.g., ABCD™) platform addresses this limitation by enabling direct loading of oligonucleotides to biopolymers. This approach preserves antibody functionality while offering flexibility in DARs. Here, we evaluated the cellular uptake and target knockdown potency of antibody biopolymer conjugate-oligonucleotides (e.g., ABCO) in the target cells, providing a proof of concept for antibody biopolymer conjugate (e.g., ABC)- mediated intracellular delivery of oligonucleotide therapeutics.

[0236] Methods: antibody biopolymer conjugate oligonucleotide (e.g., ABCO) molecules were generated by conjugating oligonucleotide payload, biopolymer, and an antitransferrin receptor 1 (TfRl) antibody. The binding affinity of anti-TfRl antibody biopolymer conjugate oligonucleotide (e.g., ABCO) was assessed using surface plasmon resonance (SPR). Cellular uptake of anti-TfRl antibody and its bioconjugates was evaluated by an antibody internalization assay using low-pH fluorescence dye. The target gene knockdown potency of antibody biopolymer conjugate oligonucleotides (e.g., ABCOs) was investigated through in vitro cell treatment, followed by RNA extraction and gene expression analysis using real-time PCR.

[0237] Results: In SPR assays, anti-TfRl antibody biopolymer conjugate oligonucleotide (e.g., ABCO) showed high binding affinity to recombinant TfRl protein. Anti- TfRl antibody, ABC control, and antibody biopolymer conjugate oligonucleotide (e.g., ABCO) exhibited concentration-dependent internalization fluorescence signal. Although oligonucleotide loading slightly reduced the cellular uptake of the anti-TfRl ABCO, gene knockdown assay with anti-TfRl antibody biopolymer conjugate oligonucleotide (e.g., ABCO) treatment demonstrated effective modulation of target gene expression.

[0238] Conclusions: Anti-TfRl antibody biopolymer conjugate oligonucleotide (e.g., ABCO)-mediated delivery of oligonucleotides and target gene knockdown demonstrate the capability of intracellular drug delivery using the antibody biopolymer conjugate drug (e.g., ABCD™) platform. With its ability to accommodate diverse drug modalities and enhance drug-loading capacity, the platform offers a promising approach for developing innovative therapies for the treatment of ocular and systemic diseases.

Claims

WHAT IS CLAIMED IS:

1. A method for delivering a payload to a cell, the method comprising: providing a cell-surface-targeting-domain-polymer-payload conjugate

(CSTDPPC) comprising: a cell -surface-targeting domain (CSTD) configured for binding to one or more targets on a surface of a cell; a copolymer conjugated to the CSTD, the copolymer comprising one or more polymer arms extending from an initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer; and one or more payloads linked to the copolymer and/or the CSTD, optionally, wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400; and administering the CSTDPPC to a cell, thereby binding the CSTDPPC to the cell, wherein the binding results in internalization thereby internalizing the one or more payloads, and wherein the internalized one or more payloads are released from the polymer.

2. The method of claim 1, wherein the first and second monomers are distributed randomly throughout the copolymer.

3. The method of claim 1, wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone.

4. The method of claim 1, wherein the one or more payloads are linked to the polymer via the second monomer.

5. The method of claim 1, wherein the CSTDPPC comprises a cleavable linker that covalently links the one or more payloads to the copolymer.

6. The method of claim 1, wherein the copolymer comprises multiple polymer arms extending from the initiator fragment.

7. The method of claim 6, wherein initiator fragment is a branched initiator fragment.

8. The method of claim 1 wherein the cell is within a subject.

9. The method of claim 1, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment that conjugates with the CSTD;

I’ is a radical scavenger, such that the combination of I-F is an initiator, I¹, for the polymerization of the copolymer of Formula I; subscripts x and y¹ are each independently an integer of from 1 to 1000; subscript z is an integer of from 1 to 10; subscript s is an integer of from 1 to 100; and subscript n is an integer of from 1 to 20.

10. The method of claim 1, wherein the copolymer is represented by Formula I: (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the CSTD or the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment;

11. The method of claim 1, wherein the copolymer is represented by Formula II: (Formula II), wherein each of R¹ and R² is H or C1-C4 alkyl, and PC is phosphorylcholine.

12. A cell-surface-targeting-domain-polymer-payload conjugate (CSTDPPC) comprising: a cell-surface-targeting domain (CSTD) configured for binding to one or more targets on a cell surface; a copolymer conjugated to the CSTD, wherein the copolymer comprises one or more polymer arms extending from an initiator fragment, each polymer arm comprising: a first monomer comprising a zwitterionic moiety; and a second monomer; and one or more payloads linked to the copolymer and/or the CSTD, optionally wherein the payload-to-cell-surface-targeting domain (“PCSTD”) ratio for at least one of the one or more payloads is between 1-400, and wherein upon binding to the target on the cell surface the CSTDPPC is internalized thereby internalizing the one or more payloads.

13. The CSTDPPC of claim 12, wherein the first and second monomers are distributed randomly throughout the copolymer.

14. The CSTDPPC of claim 12, wherein each of the first monomer and the second monomer is independently selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine and vinyl pyrrolidone.

15. The CSTDPPC of claim 12, wherein the one or more payloads are linked to the polymer via the second monomer.

16. The CSTDPPC of claim 12, wherein the CSTDPPC comprises a cleavable linker that covalently links the one or more payloads to the copolymer.

17. The CSTDPPC of claim 12, wherein the copolymer comprises multiple polymer arms extending from the initiator fragment.

18. The CSTDPPC of claim 17, wherein initiator fragment is a branched initiator fragment.

19. The CSTDPPC of claim 12, wherein the copolymer is represented by Formula I: (Formula I), each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment that conjugates with the CSTD;

20. The CSTDPPC of claim 12, wherein the copolymer is represented by Formula (Formula I), wherein each M¹ and M² are independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl pyridine and vinyl pyrrolidone;

M² links to the CSTD or the one or more payloads;

ZW is a zwitterionic moiety;

I is an initiator fragment;

21. The CSTDPPC of claim 12, wherein the copolymer is further represented by Formula II: (Formula II), wherein each of R¹ and R² is H or C1-C4 alkyl, and PC is phosphorylcholine.

22. The method of claim 1, wherein the cell-surface-targeting-domain (CSTD) is a protein selected from the group consisting of antibodies, single-chain Fvs, Fabs, single domain antibodies, centyrins, nanofitins, affibodies, DARPins, affilins, anticalins, Kunitz, avimers, monobodies, peptides, cyclic peptides, constrained peptides, trap domains, trap-domain- antibody fusions, knottins, and native and engineered proteins that to bind a target or targets on the surface of a cell.

23. The method of claim 22, wherein the cell-surface-targeting-domain is an antibody or an antigen-binding fragment thereof.

24. The method of claim 23, wherein the antibody is an anti-VEGFR2-antibody, anti-TfRl -antibody, or an anti-HER2-antibody.

25. The method of claim 1, wherein the CSTD is selected from the group consisting of aptamers, sugar moieties, macrocycles, and lipids.

26. The method of claim 25, wherein the CSTD is an aptamer, optionally wherein the aptamer targets TfRl.

27. The method of claim 1, wherein targeting is tissue specific.

28. The method of claim 1, wherein the payload is selected from the group consisting of oligonucleotides, small molecules, proteins, peptides, aptamers, macrocycles, cyclic peptides, constrained peptides, tRNAs, dye/label or combinations thereof.

29. The method of claim 28, wherein the payload is an oligonucleotide, optionally wherein the oligonucleotide is a PCSK9 siRNA or a PCSK9 antisense oligonucleotide.

30. The method of claim 28, wherein the payload is a small molecule, optionally wherein the small molecule is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

31. A cell-surface-targeting-domain-polymer-fluorescent dye (CSTDPFDC) conjugate comprising: a CSTD comprising an antibody; a polymer conjugated to the antibody; and a fluorescent dye, wherein the fluorescent dye is covalently linked to the antibody and/or the polymer.

32. The CSTDPFDC of claim 31, wherein the polymer is a homopolymer.

33. The CSTDPFDC of claim 31, wherein the polymer is a copolymer.

34. The CSTDPFDC of claim 31, wherein the fluorescent dye is covalently linked to the antibody.

35. The CSTDPFDC of claim 31, wherein the fluorescent dye is covalently linked to the polymer.

36. The method according to claim 1, wherein the polymer or copolymer has a molecular weight between about 10,000 and about 1,750,000 Da as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC-MALS”).

37. The method of claim 36, wherein the polymer or copolymer has a molecular weight of about 10,000 Da, or about 25,000 Da, or about 38,000 Da.

38. The method of claim 36, wherein the polymer or copolymer has a molecular weight of about 50,000 Da.

39. The method of claim 36, wherein the polymer or copolymer has a molecular weight of about 100,000 Da.

40. The method of claim 36, wherein the polymer or copolymer has a molecular weight of about 150,000 Da, or about 180,000 Da, or about 290,000 Da.

41. The method of claim 36, wherein the polymer or copolymer has a molecular weight of about 250,000 Da.

42. The method of claim 36, wherein the molecular weight of the polymer or copolymer is 300,000 Da or greater.

43. The method of claim 1, wherein the polymer or copolymer has a molecular weight between about 500,000 and about 1,000,000 Da.

44. The method of any one of the preceding claims, wherein the polymer or copolymer has a molecular weight between about 750,000 to about 850,000 Da.

45. The method of claim 1, wherein the PCSTD ratio is between 5-400.

46. The method of claim 1, wherein the PCSTD ratio is between 10-400.

47. The method of claim 1, wherein the PCSTD ratio is between 20-400.