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WO2025111600A1 - Compositions and methods for lipidated peptide-based modulators of hla-e - Google Patents

Compositions and methods for lipidated peptide-based modulators of hla-e Download PDF

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Publication number
WO2025111600A1
WO2025111600A1 PCT/US2024/057189 US2024057189W WO2025111600A1 WO 2025111600 A1 WO2025111600 A1 WO 2025111600A1 US 2024057189 W US2024057189 W US 2024057189W WO 2025111600 A1 WO2025111600 A1 WO 2025111600A1
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Prior art keywords
substituted
synthetic peptide
unsubstituted
hla
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2024/057189
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French (fr)
Inventor
Magdalena Preciado LOPEZ
Dan L. Eaton
Clint REMARCIK
John C.K. WANG
Nathalie M. GROB
Simon L. RÖSSLER
Anthony J. QUARTARARO
Jason TAO
Andrei LOAS
Stephen L. Buchwald
Bradley L. Pentelute
Ying Kit Jeffrey Wong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Institute of Technology
Calico Life Sciences LLC
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Massachusetts Institute of Technology
Calico Life Sciences LLC
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Publication of WO2025111600A1 publication Critical patent/WO2025111600A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • NK cells Natural Killer cells
  • T cells play an important role in the innate and adaptive immune response and in the prevention of cancer. These cells provide an efficient immunosurveillance mechanism by which undesired cells such as tumor cells or virally infected cells can be eliminated.
  • NK cell and T cell activity is regulated by a complex mechanism that involves both activating and inhibitory signals.
  • the inhibitory NK cell receptor dimer CD94/NKG2A C-type lectin receptor complex has recently been identified as an immune checkpoint in the tumor microenvironment and is expressed on NK cells as well as some T cell subsets.
  • HLA-E human leukocyte antigen E
  • High levels of HLA-E expression at the tumor cell surface are reported in several cancer types, including gynecologic cancers (up to 90% of tumor samples, e.g., cervical and ovarian) and up to 50% in breast cancer, non–small cell lung carcinoma (NSCLC), melanoma, glioblastoma, liver, pancreas, kidney, melanoma, prostate, head and neck, stomach, rectal, and colorectal cancer.
  • NSCLC non–small cell lung carcinoma
  • melanoma glioblastoma
  • liver pancreas
  • kidney melanoma
  • prostate head and neck
  • stomach rectal
  • colorectal cancer Blocking of the CD94/NKG2A receptor on NK and T cells has been shown to increase cytotoxic activity of NK and T cells.
  • the overexpression of HLA-E by cancer cells is a potential strategy to escape immune surveillance and protect cancer cells from NK cell-mediated cytolytic attacks.
  • the present disclosure is directed, at least in part, to synthetic peptides, peptidomimetics, compositions, and methods for the modulation of HLA-E-CD94/NKG2A interaction (e.g., activation of CD94/NKG2A signaling).
  • the present disclosure provides a synthetic peptide comprising an amino acid sequence X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3), wherein each of X 1 , X 3 , X 4 , and X 7 are independently selected from 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg; X 5 is Ala, Cha, Tha, or Mff; and X 8 is Arg, Msn, or hAr; wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the linker connects the C18 octadecanedioic acid to the Lys residue.
  • the amino acid sequence is (a) NH 2 -4Af-Met-Dff-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 5); (b) NH 2 -hAr-Met-Msn-Dff-Cha-Ala-Arg-Msn-Leu-OH (SEQ ID NO: 6); (c) NH 2 -Ala-Met-hAr-Dff-Tha-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 7); (d) NH 2 -4Af-Met-Ala-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 8); or (e) NH 2 -hAr-Met-Har-Gln-M
  • one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 and is substituted with a Cys.
  • the one or more Cys, Lys, Tyr, His, Ser, or Thr is arylated.
  • the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead.
  • the one or more other amino acids is substituted with a Lys and is conjugated to a warhead, wherein conjugation is to the N atom on the side chain.
  • the one or more other amino acids is 2 IPTS/128777599.4 Attorney Docket No.: CLS-039WO substituted with a Tyr, Ser, or Thr and is conjugated to a warhead, wherein conjugation is to the O atom on the side chain.
  • the one or more other amino acids is substituted with a His and is conjugated to a warhead, wherein conjugation is to the N atom on the side chain.
  • the warhead is , wherein X is a halogen. [0015] In some embodiments, the warhead is . [0016] In some embodiments, the warhead is selected from the group consisting of 5 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0017] In some embodiments, the warhead is selected from the group of a sulfonyl fluoride, a phenyl carbamate, and a squaramate. [0018] In some embodiments, the warhead is conjugated to the Cys via the sulfur atom of the Cys.
  • the present disclosure also provides a synthetic peptide comprising an amino acid sequence hAr-X 2 -hAr-Gln-Mff-A-Cha-hAr-X 9 (SEQ ID NO: 20) wherein X 2 is Nle or Mox; and X 9 is Leu, Aoa, or Cha; wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the amino acid sequence is “ (a) NH 2 -hAr-Nle-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (“B9”; SEQ ID NO: 21); (b) NH2-hAr-Mox-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (“B10”; SEQ ID NO: 22); (c) NH 2 -hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Nle-OH (“B6”; SEQ ID NO: 23); (d) NH 2 -hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Aoa-OH (“B7”; SEQ ID NO: 24); or (e) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Leu-
  • the amino acid sequence is NH 2 -hAr-Nle-hAr-Gln-Mff-Ala-Cha- hAr-Leu-OH (SEQ ID NO: 21).
  • the Lys-R y substitution is at position 5. 6 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
  • one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys.
  • the present disclosure provides a synthetic peptide comprising the formula wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising an amino acid sequence VMAPRT(L/V)(V/L/I/F)L, wherein one or more amino acids is substituted with a Lys-R y ; wherein the R y comprises C18 octadecanedioic acid and a linker; and wherein one or more other amino acids are substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8.
  • the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a cysteine.
  • the peptide has the amino acid sequence VMAPRTLFL.
  • the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead.
  • the warhead is , wherein X is a halogen. [0032] In some embodiments, the warhead is . [0033] In some embodiments, the warhead is selected from the group consisting of 11 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0034] In some embodiments, the warhead is conjugated to the Cys via the sulfur atom of the Cys. [0035] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula , [0036] wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula 12 IPTS/128777599.4 Attorney Docket No.: CLS-039WO , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , 13 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , 14 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker [0043] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula 15 IPTS/128777599.4 Attorney Docket No.: CLS-039WO , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula O F , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , 16 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula O 17 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the present disclosure provides a synthetic peptide comprising the formula F S O O , wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker.
  • the synthetic peptide is an HLA-E-NKG2A complex specific inhibitor.
  • the present disclosure provides a synthetic peptide comprising the amino acid sequence of VMAPRTLFL, wherein one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises C18 octadecanedioic acid and a linker; and further comprising one or more other amino acid substitution,
  • the one or more other amino acid substitution comprises: a. a substitution of the V residue at a position 1; b.
  • the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg.
  • the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAr. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr.
  • the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. 19 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0055]
  • the linker comprises a PEG linker.
  • the linker comprises a PEG2 linker.
  • the linker comprises two PEG2 linkers.
  • the PEG2 linker is 8-amino-3,6-dioxaoctanoic acid.
  • the linker comprises a ⁇ -Glu.
  • the present disclosure provides a synthetic peptide comprising the formula [0057]
  • the present disclosure provides a synthetic peptide as disclosed herein comprising one or more additional modifications selected from: a. acetylated, formylated, propanoylated, hexanoylated, or myristoylated N-terminus; b. amidated C-terminus; c. substitution of one or more L-amino acid with a D-amino acid; d. substitution of one or more amino acid with a methyl-amino acid; and e. substitution of an ⁇ -amino acid with a ⁇ -amino acids.
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein.
  • the present disclosure provides a peptide or peptidomimetic comprising the amino acid sequence of any one of Tables 3-19. 20 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0059]
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein, comprising one or more amino acid substitutions for reducing interaction of HLA-E with CD94/NKG2A, optionally relative to a comparator or the unmodified counterpart.
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein that reduces interaction of HLA-E with CD94/NKG2A, optionally relative to a comparator or the unmodified counterpart.
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein, comprising one or more amino acid substitutions for increasing NK-mediated cell killing, optionally as measured by a luciferase- based assay, optionally relative to a comparator or the unmodified counterpart.
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein that increases NK-mediated cell killing, optionally as measured by a luciferase-based assay, optionally relative to a comparator or the unmodified counterpart.
  • the present disclosure provides a peptide or peptidomimetic as disclosed herein, lipidated to increase stability, optionally relative to a comparator or the unmodified counterpart.
  • a synthetic peptide or peptidomimetic as disclosed herein comprises an alanine substitute at position 1 or position 7.
  • a synthetic peptide or peptidomimetic as disclosed herein capable of displacing VL9 or VL9*5FAM from the antigen-binding groove of HLA-E.
  • the synthetic peptide or peptidomimetic has higher displacement efficiency at the antigen-binding groove of HLA-E relative to a comparator or the unmodified counterpart, optionally as analyzed by LC-MS or fluorescence polarization assays.
  • the present disclosure provides a synthetic peptide/HLA-E complex, wherein the peptide is selected from a synthetic peptide as disclosed herein.
  • the synthetic peptide and the HLA-E in the complex are covalently linked.
  • the HLA-E is human HLA-E.
  • the synthetic peptide is covalently linked to amino acid residue Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E.
  • the synthetic peptide is covalently linked to an amino acid residue selected from the group of Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln- 72, Asn-77, Thr-80, Tyr-84, Trp-97, His-99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser- 147, Glu-152, His-155, Gln-156, Tyr-159, Thr-163, Cys-164, Trp-167, and Tyr-171 of human HLA-E.
  • FIG. 1A depicts HLA-E loaded with VL9 on cancer cells binding to CD94–NKG2A on NK cells and preventing a cytolytic attack by the NK cell.
  • FIG. 1B shows a top view of VL9 binding the groove formed by two ⁇ -helices of HLA-E. Met-2 and Leu-9 anchor the 9-mer peptide in the binding groove.
  • FIG. 1C depicts the design of a mixed 9- and 10-mer library based on VL9 including three different sets of amino acids.
  • FIG. 1D depicts non-canonical amino acids used in the design of a combinatorial library.
  • FIGs. 2A-2F show the chemical formulas for peptides B1-B5 (SEQ ID NOs: 5-9) and B5 scrambled.
  • FIG. 2G shows hit peptides B1–B5 containing positively charged residues (*) at the C- and N-termini and hydrophobic residues (#) in the core region.
  • FIGs. 3A-3F show the chemical formulas for peptides B9 to B8 and a scrambled control peptide.
  • FIG. 4 illustrates the results of a peptide exchange experiment of HLA-E/VL9 with exemplary peptides B1, B2, B3, B4, B5, B9, B10, B6, B7 and B8.
  • FIG. 5A shows peptides B6–B10. Nle, Aoa, and Cha represent alternative amino acids of aliphatic Leu, whereas Nle and Mox were used to substitute the oxidation-sensitive thioether in Met.
  • FIG. 5B shows some non-natural amino acids that can be single substitutions of Met2 or Leu9.
  • FIG. 5C illustrates the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B1, B2, B3, B4, B5, and B9).
  • FIG. 5D depicts the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B5, B9, B10, B6, B7 and B8).
  • FIG. 5E depicts the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B5, B9, B5scrambled).
  • FIG. 5F shows that all analogs of B5 displayed a high degree of exchange with HLA-E (low VL9 abundance in 22 IPTS/128777599.4 Attorney Docket No.: CLS-039WO intact HLA-E).
  • FIG. 5G shows Peptides B6–B10 showed potent inhibition of CD94–NKG2A binding by BLI, whereas B9 scrambled displayed no inhibition.
  • FIG. 5H shows Peptides B6– B10 induced loss of fluorescence polarization (measured in mP) over 4 h, whereas B9 scrambled and DMSO displayed no change of fluorescence polarization.
  • FIG. 5I includes a plot depicting the thermal stability of the HLA-E/B2M complex loaded with the indicated peptides.
  • FIG. 5J shows the melting temperature (Tm) shift for indicated alanine-scan peptide derivatives of B9 relative to B9.
  • FIG. 6A illustrates a chemical reaction for installing an electrophilic aryl sulfonyl fluoride warhead by Pd-mediated coupling using an oxidative addition complex.
  • FIG. 6B illustrates a reaction for cross-linking of electrophilic analogs of VL9 to HLA-E.
  • FIG. 6C illustrates the results for exemplary cross-linking reactions with the electrophilic analogs of VL9 as measured my Mass Spectrometry (MS).
  • MS Mass Spectrometry
  • FIG. 6D illustrates nucleophilic residues in a crystal structure on HLA-E situated within 6 ⁇ from the binding groove of VL9 (highlighted in yellow).
  • FIG. 7 illustrates an exemplary synthesis of Palladium Oxidative Addition Complex, (RuPhos)Pd(m-benzenefluorosulfonyl)Br, 1.
  • FIG. 8A illustrates an exemplary peptide with a warhead (B9_8*) that is a covalent binder selective for HLA-E and inhibits binding of CD94/NKG2A. B9 was equipped with an electrophilic warhead at position 8 for covalent binding.
  • FIG. 8B illustrates the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B9_8* and VL- 9_8*).
  • FIG. 8C shows the results of crosslinking experiments measured MS of exemplary peptide/HLA-E complexes (grey traces represent protein reference spectra prior to incubation).
  • FIG. 9 illustrates a synthesis schematic for making a candidate lipidated peptide.
  • FIGs. 10A and 10B illustrate the structure, physical properties, and chromatogram results for the indicated lipidated peptide and its scrambled control, respectively.
  • FIG. 10A and 10B illustrate the structure, physical properties, and chromatogram results for the indicated lipidated peptide and its scrambled control, respectively.
  • FIG. 11 illustrates the LC-MS plots from B9 and lipidated B9 were incubated in 25% human serum or 10% FBS for 24 hours, showing that the lipidation increases B9’s stability in serum-containing media.
  • FIG. 12 illustrates the indicated lipidated B9 peptide and control exchange rates with VL9. The exchange rate of B9 is not impaired by lipidation in PBS, and is enhanced in FBS- containing media relative to B9.
  • FIG. 13 illustrates the indicated lipidated B9 peptide and control B9 BLI inhibition are similar in PBS, but lipidated B9 outperforms B9 in FBS-containing media. 23 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0078] FIGs.
  • FIGS. 14A-14G show the interaction of HLA-E with CD94–NKG2A can be inhibited by peptides discovered from a focused library.
  • FIGs. 14A-14B show how HLA-E/B2M/VL9 (1 ⁇ M) was incubated with 10-fold excess of hit peptidomimetics (10 ⁇ M) for ligand exchange and subsequently loaded onto biosensors to measure association and dissociation of CD94–NKG2A (200 nM).
  • FIG. 14C shows the binding of CD94–NKG2A was inhibited by peptides B2, B3, B5, after incubation with HLA-E as determined by BLI.
  • FIG. 14D shows how the abundance of VL9 on intact HLA-E complexes was determined by LC-MS after ligand exchange.
  • FIG. 14E shows VL9 abundance was the lowest after B5 exchange for 16 h at room temperature.
  • FIG. 14F shows how the displacement of fluorescently labeled VL9*5FAM from the complex with HLA- E and B2M by the hit sequences was monitored by changes in fluorescence polarization ( ⁇ mP).
  • FIG. 14G shows Peptides B1, B2, B3, and B5 led to changes in fluorescence polarization (measured in mP) over 4 h.
  • FIGs. 15A-15F show the crystal structure of HLA-E/B2M/B9.
  • FIG. 15A Overall structure.
  • HLA-E and B2M are shown as surface and cartoon representations and B9 is shown as sticks.
  • FIG. 15B Detailed interactions between HLA-E and B9. Arg-62 from the NKG2A/CD94/HLA-E/B2M/VL9 co-receptor structure is shown as transparent green sticks. Hydrogen bonds are shown as black, dashed lines.
  • FIG. 15C Alignment of the B9-bound HLA- E/B2M structure (with only B9 peptide shown) with the NKG2A/CD94/HLA-E/B2M/VL9 co- receptor structure (with only protein shown) reveals several clashes.
  • FIG. 15C Alignment of the B9-bound HLA- E/B2M structure (with only B9 peptide shown) with the NKG2A/CD94/HLA-E/B2M/VL9 co- receptor structure (with only protein shown) reveals several clashes.
  • FIG. 15C Alignment of the B9-bound
  • FIGs. 16A-16H show that B9 outperforms Mtb44 (a known inhibitory peptide) in biophysical assays.
  • FIG. 16A Exchange of B9 with HLA-E/VL9*5FAM was compared to Mtb44 with a higher degree of VL9 exchange via LC-MS.
  • FIG. 16B Validation of exchange was shown through loss of polarization with VL9, B9, and Mtb44 showing similar exchange kinetics in comparison to their scrambled counterparts.
  • FIG. 16A Exchange of B9 with HLA-E/VL9*5FAM was compared to Mtb44 with a higher degree of VL9 exchange via LC-MS.
  • FIG. 16B Validation of exchange was shown through loss of polarization with VL9, B9, and Mtb44 showing similar exchange kinetics in comparison to their scrambled counterparts.
  • FIG. 16C BLI demonstrated a stronger loss of binding with CD94-NKG2A than Mtb44.
  • FIG. 16D B9, VL9, Mtb44 or scrambled analogs thereof bearing a fluorescent label were incubated with HLA-R/B2M/VL9 to monitor changes of FP over 2 h indicating binding of the fluorescent peptide to HLA-E.
  • FIG. 16H The strongest gain of FP was observed for B9*5FAM, whereas the scrambled sequence analogs showed no change of FP over 2 h.
  • FIGs. 17A-17F shows inhibitory peptide B9 led to HLA-E stabilization and loss of CD94-NKG2A binding on cells.
  • FIG. 17A-17F shows inhibitory peptide B9 led to HLA-E stabilization and loss of CD94-NKG2A binding on cells.
  • FIG. 17A HLA-E (3D12-APC) staining of SCaBER cells after VL9 pulse and peptide exchange. Plotted medians on the graph to the right of the histograms.
  • FIG. 17B HLA-E/VL9 (tetramerized CD94-NKG2A/StrepAF647) staining of SCaBER cells 24 IPTS/128777599.4 Attorney Docket No.: CLS-039WO after VL9 pulse and peptide exchange. Plotted medians on the graph to the right of the histograms.
  • FIG. 17C HLA-E (3D12-APC) staining of the competition of peptides with VL9 at ratiometric levels (total peptide concentration of 20uM).
  • FIG. 17D HLA-E/VL9 (tetramerized CD94-NKG2A/StrepAF647) staining of the competition of peptides with VL9 at ratiometric levels (total peptide concentration of 20uM).
  • FIG. 17E Jurkat reporter stimulation after pulse and exchange with peptides at 10uM and 20uM exchanged peptides. Luminescence was measured as a report on Jurkat interaction with cells. Comparison to surface levels of HLA-E stained with 3D12-APC and analyzed via FACS.
  • FIG. 17E HLA-E/VL9 (tetramerized CD94-NKG2A/StrepAF647) staining of the competition of peptides with VL9 at ratiometric levels (total peptide concentration of 20uM).
  • FIG. 17E Jurkat reporter stimulation after pulse and exchange with peptides at 10uM and 20uM exchanged peptides. Luminescence was measured as a report on Jurkat interaction with cells. Comparison to
  • 17F shows Lucia® luciferase secretion, as measured in NFAT-Lucia reporter system, for VL9, B9, and Mtb44 groups.
  • the present disclosure is based, in part, upon the development of synthetic peptides and peptidomimetics that bind HLA-E in a covalent or non-covalent manner to form peptide-HLA-E complexes. Additionally, the peptide-HLA-E complexes can modulate or inhibit the binding of HLA-E to its cognate receptor CD94/NKG2A or prevent activation of CD94/NKG2A.
  • compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present invention that consist essentially of, or consist of, the recited 25 IPTS/128777599.4 Attorney Docket No.: CLS-039WO components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40
  • an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56, and/or CD57, the absence of the alpha/beta or gamma/delta T- 27 IPTS/128777599.4 Attorney Docket No.: CLS-039WO cell receptor (TCR) complex on the cell surface, the ability to bind to and kill cells that fail to express “self MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK-activating receptors, and the ability to release cytokines that stimulate or inhibit the immune response.
  • TCR CLS-039WO cell receptor
  • NKG2A or “KLRC1” refers to the full length NKG2A.
  • NKG2A OMIM 161555, the entire disclosure of which is herein incorporated by reference
  • NKG2A is a member of the NKG2 group of transcripts (see e.g., Houchins, et al. (1991), The Journal of experimental medicine).
  • NKG2A and NKG2C form heterodimer receptors with CD94 and both target the same p/HLA-E complex, but ligation with the p/HLA-E complex induces an inhibitory signal for NKG2A and an activation signal for NKG2C.
  • HLA-E presents a limited set of conserved signal peptides to NKG2A and NKG2C. These peptides bound and presented by HLA-E are derived from leader sequences of classical HLA class I molecules.
  • HLA-E human leukocyte antigen-E
  • the peptide–HLA-E complex is expressed in most human tissues as a marker of cell health and protects cells from the cytotoxic activation of NK cells.
  • the expression of HLA-E has also been associated with different types of cancer as a mechanism to evade attacks by NK cells.
  • MHC major histocompatibility complex
  • MHC class I or MHC I are used interchangeably to refer to protein molecules comprising an ⁇ chain composed of three domains ( ⁇ 1, ⁇ 2 and ⁇ 3), and a second, invariant ⁇ 2-microglobulin.
  • the ⁇ 3 domain is linked to the transmembrane domain, anchoring the MHC class I molecule to the cell membrane.
  • Antigen-derived peptide epitopes which are located in the peptide-binding groove, in the central region of the ⁇ 1/ ⁇ 2 heterodimer.
  • MHC Class I molecules such as HLA-A, HLA-B, HLA-C, and HLA-E are part of a process that presents short polypeptides to the immune system. These polypeptides are typically 8-11 amino acids in length and originate from proteins being expressed by the cell, which can be endogenous proteins or exogenous proteins (e.g., viral or bacterial proteins, vaccine proteins).
  • Histocompatibility leucocyte antigen E (HLA-E) is a conserved nonclassical HLA class I molecule that binds a limited peptide repertoire.
  • Antigens delivered endogenously to APCs are processed primarily for association with MHC class I.
  • Antigens delivered exogenously to APCs are processed primarily for association with MHC class II.
  • MHC proteins also includes MHC variants which contain amino acid substitutions, deletions or insertions and yet which still bind MHC peptide epitopes (MHC Class I or MHC Class II peptide epitopes).
  • MHC MHC molecule
  • MHC protein also includes an extracellular fragment of a full-length MHC protein that retains the ability to bind the cognate epitope, for example, a soluble MHC.
  • soluble MHC refers to an extracellular fragment of a MHC comprising corresponding ⁇ 1 and ⁇ 2 domains that bind a class I T cell epitope or corresponding ⁇ 1 and ⁇ 1 domains that bind a class II T cell epitope, where the ⁇ 1 and ⁇ 2 domains or the ⁇ 1 and ⁇ 1 domains are derived from a naturally occurring MHC or a variant thereof.
  • the classical MHC class I (termed “Ia”) molecules HLA-A, HLA-B and HLA-C
  • HLA-A, HLA-B and HLA-C are highly polymorphic and are ubiquitously expressed on most somatic cells.
  • HLA-E non classical MHC class I (termed “Ib”) molecules
  • HLA-F HLA-F
  • HLA-G non classical MHC class I molecules
  • HLA-E refers wild type, full length HLA-E.
  • class Ib molecules HLA-E is characterized by a low polymorphism and a broad mRNA expression on different cell types.
  • HLA-E is nonpolymorphic with only two functional alleles present in the human population: the HLA-E*01:01 and the HLA-E*01:03 variants.
  • This class I molecule is a heterodimer consisting of a heavy chain and a light chain ( ⁇ 2-microglobulin, ⁇ 2m, B2M).
  • the heavy chain is approximately 45 kDa and its gene contains 8 exons.
  • Cell surface expression of HLA-E requires the availability of ⁇ 2- microglobulin (Ulbrecht et al. (1999) Eur J Immunol.
  • HLA-E binds NK cells and some T cells, binding specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C, and not to the inhibitory KIR receptors. See, e.g., Braud et al. (1998) Nature 391 :795-799.
  • MHC protein also includes MHC proteins of non-human species of vertebrates.
  • MHC proteins of non-human species of vertebrates play a role in the examination and healing of diseases of these species of vertebrates, for example, in veterinary medicine and in animal tests in which human diseases are examined on an animal model, for example, experimental autoimmune encephalomyelitis (EAE) in mice (mus musculus), which is an animal model of the human disease multiple sclerosis.
  • EAE experimental autoimmune encephalomyelitis
  • Non-human species of vertebrates are, for example, and more specifically mice (mus musculus), rats (rattus norvegicus), cows (bos taurus), horses (equus equus) and green monkeys (macaca mulatta).
  • MHC proteins of mice are, for example, referred to as H-2-proteins, wherein the MHC class I proteins are encoded by the gene loci H2K, H2L, and H2D and the MHC class II proteins are encoded by the gene loci H2I.
  • the term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • expression vectors are referred to herein as “expression vectors.”
  • the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom.
  • Such progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations.
  • the term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.
  • modulation refers to an increase or decrease in the level of a target molecule or the function of a target molecule.
  • modulator refers to 30 IPTS/128777599.4 Attorney Docket No.: CLS-039WO modulation of (e.g., an increase or decrease in) the level of a target molecule or the function of a target molecule.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw–Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).
  • HPLC high pressure liquid chromatography
  • the invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
  • 31 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
  • k d refers to the dissociation rate constant between a given entity and a target (e.g., of a particular peptide-target interaction). This value is also referred to as the k off value.
  • k a (M -1 ⁇ s -1 ) refers to the association rate constant of a given entity and a target (e.g., a particular peptide-target interaction).
  • K D K D
  • K D k d /k
  • lipidated refers to a synthetic peptide or peptidomimetic comprising a lipid moiety (e.g., C18 octadecanedioic acid group).
  • lipidation of peptides can be used to increase, for example, the pharmacokinetic properties, improve metabolic stability, reduce enzymatic degradation, lower excretion and metabolism, in vivo half-life, etc.
  • the terms “B5.1” and “B9” are used interchangeably.
  • the terms “B5.2” and “B10” are used interchangeably.
  • the terms “B5.3” and “B6” are used interchangeably.
  • the terms “B5.4” and “B7” are used interchangeably.
  • a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
  • an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, 32 IPTS/128777599.4 Attorney Docket No.: CLS-039WO in enantiomeric excess of the “R” form.
  • enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer.
  • the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
  • an enantiomerically pure compound can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising enantiomerically pure R–compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R–compound.
  • the enantiomerically pure R– compound in such compositions can, for example, comprise, at least about 95% by weight R– compound and at most about 5% by weight S–compound, by total weight of the compound.
  • a pharmaceutical composition comprising enantiomerically pure S–compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S–compound.
  • the enantiomerically pure S– compound in such compositions can, for example, comprise, at least about 95% by weight S– compound and at most about 5% by weight R–compound, by total weight of the compound.
  • the active ingredient can be formulated with little or no excipient or carrier. [0120] When a range of values is listed, it is intended to encompass each value and sub–range within the range.
  • C1-C6 alkyl is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4- C5, and C5-C6 alkyl.
  • Aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”).
  • an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1– naphthyl and 2–naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl).
  • An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
  • Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
  • Each 33 IPTS/128777599.4 Attorney Docket No.: CLS-039WO instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is unsubstituted C6-C14 aryl.
  • the aryl group is substituted C6-C14 aryl.
  • Halo or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.
  • halide by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom. In certain embodiments, the halo group is either fluorine or chlorine.
  • linker refers to either a bond or a moiety which at one end exhibits a grouping able to enter into a covalent bonding with the residue or a reactive functional group of the synthetic peptide or peptidomimetic, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding, and bridges, either directly or indirectly the synthetic peptide or peptidomimetic and the lipid moeity, e.g. C18 octadecanedioic acid group.
  • spacer and linker interchangeably.
  • the linker is at least one polyethylene glycol polymer.
  • the linker attached to a Lysine residue in the peptide or peptidomimetic.
  • the linker can be at a primary amine (-NH-) 2 present at the N-terminus of each polypeptide chain (referred to as ⁇ -amine) or the side chain of the lysine (Lys, K) residue (referred to as E-amine).
  • a linker can be attached to a peptide compound at a sulfhydryl group (-SH): this group is present in the side chain of cysteine (Cys, C).
  • cysteines are linked together between their side chains by disulfide bonds (- S-S-) as part of the secondary or tertiary structure of the protein. These must be reduced to a thiol group to make them available for most types of reactive groups to crosslink.
  • the linker can be attached to the peptide compound at a carbonyl (-CHO): by oxidation of post-translational modifications (glycosylation) of polysaccharides with sodium metaperiodate, ketone or aldehyde groups can be generated in glycoproteins.
  • the linker can be a cleavable linker.
  • the linker can be a non-cleavable linker.
  • polyethylene glycol refers to a polymer containing ethylene glycol monomer units of formula —O—CH2—CH2—.
  • suitable polyethylene glycols may have a free hydroxy group at each end of the polymer molecule or may have one hydroxy group etherified with a lower alkyl, e.g., a methyl group.
  • derivatives of polyethylene glycols having esterifiable carboxy groups are also suitable.
  • HLA-E Human leukocyte antigen E
  • MHC non-classical major histocompatibility complex
  • HLA-E displays limited polymorphism with two dominant alleles, HLA-E*01:01 and HLA-E*01:03, differing by only one amino acid which are detected in over 99% of the population.
  • HLA-E in complex with beta-2 microglobulin (B2M), is expressed on the cell surface of nucleated cells with broad tissue distribution and typically low expression levels(Wei, X., and Orr, H. T. (1990) Human Immunology). Its primary function is the presentation of antigens for immune surveillance (Braud, V. M. et al.
  • HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A, Proceedings of the National Academy of Sciences ). In homeostasis, HLA-E binds signal peptides derived from the leader sequences of MHC class I molecules HLA-A, B, C, or G as a sign of cellular health.
  • VL9 These signal peptides are collectively referred to as “VL9,” and are nonameric peptides of the general sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1).
  • VMAPRT L/V
  • SEQ ID NO: 1 The HLA-E/B2M/VL9 complex is recognized by the inhibitory 35 IPTS/128777599.4 Attorney Docket No.: CLS-039WO receptor heterodimer CD94–NKG2A of natural killer (NK) cells to prevent a cytolytic response (FIG.
  • HLA-E receptors have been shown to present other foreign and stress-related antigens to ⁇ receptors on CD8+ T-cells.
  • pathogens e.g., cytomegalovirus
  • HLA-E-restricted CD8+ T-cells represent a considerable component of the adaptive immune response, highlighting the potential of this protein target for vaccine development (Walters, L. C., et al. (2016) Nature Communications; Voogd, L., et al. (2022) Trends in Immunology; Hansen, S. G.et al. (2016) Science; Hansen, S. G., et al. (2013) Science).
  • the inventors leveraged available structural and functional information for the discovery of non-natural peptide ligands that retain binding to HLA-E but abrogate interactions with CD94–NKG2A, in part, through the use of affinity selection–mass spectrometry (AS-MS).
  • AS-MS affinity selection–mass spectrometry
  • the peptide or peptidomimetic has the amino acid sequence of an antigen.
  • Peptide antigens comprise, but are not limited to peptides that have the amino acid sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1), ( “VL9”), derived from signal peptides of the MHC class I molecules HLA-A, -B, -C, and -G.
  • the peptide or peptidomimetic is based on the ligand for the NKG2A/CD94 inhibitory receptor in mice, the nonclassical MHC molecule Qa-1b, the mouse HLA-E ortholog, which presents the peptide AMAPRTLLL (SEQ ID NO: 355), referred to as “Qdm” (for Qa-1 determinant modifier).
  • a comparator is fluorescent analog of VL9 bearing a fluorescein at position 5 (i.e., VL9*5FAM).
  • a comparator is Mtb44.
  • Mtb44 (having the amino acid sequence of RLPAKAPLL) is a peptide derived from the pathogenic Mycobacterium tuberculosis. Mtb44 is a well-characterized binder of HLA-E with comparable binding affinity as endogenous VL9 and has demonstrated ability to inhibit coreceptor interaction.
  • the peptide sequence comprises the amino acid sequence VMAPRTLVL (SEQ ID NO: 2). In some embodiments, the peptide is 8, 9, or 10 amino acids long. In some embodiments one or more amino acids of the VL9 sequence are substituted.
  • the substitution is a substitution of the V residue at position 1 (Val1), the M 36 IPTS/128777599.4 Attorney Docket No.: CLS-039WO residue at position 2 (Met2), the A residue at position 3 (Ala3), P residue at position 4 (Pro4), R residue at position 5 (Arg5), the T residue at position 6 (Thr6), the L residue at position 7 (Leu7), the F residue at position 8 (Phe8), the L residue at position 9 (Leu9), or a combination of any of the foregoing substitutions.
  • the anchor residues Met2 and Leu9 are constant.
  • the residue at position 10 is a Lys.
  • the R residue at position 5 is substituted with aliphatic and aromatic monomers
  • the F residue at position 8 is substituted by polar and charged residues.
  • the peptide antigen comprises, but are not limited to peptides that have the amino acid sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1), (referred to as VL9), derived from signal peptides of the MHC class I molecules HLA-A, -B, -C, and -G.
  • the substituted amino acid is a canonical amino acid. Canonical amino acids for use in substitutions are listed in TABLE 1.
  • the canonical substituted amino acids are an Ala, a Ser, a Gln, or an Arg.
  • TABLE 1 Canonical amino acids used in the peptides and peptidomimetics.
  • the synthetic peptide or peptidomimetic comprises one or more of 43 non-canonical amino acids.
  • amino acids in VL9 are 37 IPTS/128777599.4 Attorney Docket No.: CLS-039WO substituted with non-canonical amino acids.
  • Non-canonical amino acids that can be used for substitution are shown in TABLE 2.
  • TABLE 2 Non-canonical amino acids for use in the peptides and peptidomimetics.
  • substitutions in the VL9 peptide comprise the non-canonical amino acids 4Af, hAr, Dff, Msn, or Cha.
  • the amino acids of the synthetic peptide are mixed canonical and non-canonical amino acids.
  • hAr and hArg are used interchangeably.
  • the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg.
  • the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg.
  • the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAR. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr.
  • the amino acid sequence of the peptide or peptidomimetic comprises X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3) or X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 - Leu-Lys (SEQ ID NO: 4), wherein each of the X 1 , X 3 , X 4 , and X 7 are independently selected from Gly, Ala, Met, Pro, Cpa, Cha, Ser, Asn, Gln, Msn, Phe, Tyr, His, Trp, 4Py, 4Af, Tha, Dff, Asp, Glu, Lys, Arg, hAr, or Aad; X 5 is Gly, Ala, Val, Leu, Met, Pro, Cpa, Cba, Cha, Aoa, Phe, Trp, Mf
  • the amino acid sequence of the peptide or peptidomimetic comprises X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3) or X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 - 39 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Leu-Lys (SEQ ID NO: 4), wherein each of X 1 , X 3 , X 4 , and X 7 are independently selected from an amino acid of mixed polarity.
  • the amino acid sequence of the peptide or peptidomimetic comprises X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3) or X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 - Leu-Lys (SEQ ID NO: 4), wherein X 5 is an apolar amino acid.
  • the amino acid sequence of the peptide or peptidomimetic comprises X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3) or X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 - Leu-Lys (SEQ ID NO: 4), wherein X 8 is a polar amino acid.
  • the amino acid sequence of the peptide or peptidomimetic comprises a modified side chain. Exemplary side chains that can be used to modify the peptide or peptidomimetic of the disclosure are listed in TABLE A.
  • amino acid sequence of the peptide or peptidomimetic is selected form the amino acid sequences in TABLE 3.
  • the substituted peptide is selected from the group of SEQ ID NOs: 5-9.
  • additional amino acids in SEQ ID NOs: 3-9 are substituted.
  • one or more amino acid is substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the substituted amino acid is at position 3 or position 8.
  • the substituted amino acid is at position 8 and is substituted with a Cys. In some embodiments, the substituted amino acid is at position 3 and is substituted with a Cys. Exemplary Cys substituted peptides and peptidomimetic sequences are listed in TABLE 4. In some embodiments, the Cys substituted peptide is selected from the group of SEQ ID NOs: 10- 19. [0149] In some embodiments, terminal NH 2 - refers to the N-terminus within the adjacent listed amino acid. For example, the “NH 2 -” in “NH 2 -Ala-” refers to N-terminus within the alanine residue.
  • a terminal “-OH” or “-COOH” refers to the C-terminus within the adjacent listed amino acid.
  • the “-OH” in “Leu-OH” refers to the C-terminus within the leucine residue.
  • the amino acid sequence of the peptide or peptidomimetic comprises hAr-X 2 -hAr-Gln-Mff-A-Cha-hAr-X 9 (SEQ ID NO: 20) wherein X 2 is Nle or Mox; and X 9 is Leu, Aoa, or Cha.
  • SEQ ID NO: 20 hAr-X 2 -hAr-Gln-Mff-A-Cha-hAr-X 9
  • X 2 is Nle or Mox
  • X 9 is Leu, Aoa, or Cha.
  • TABLE 5 exemplary amino acid sequences of peptidomimetics disclosed herein.
  • additional amino acids in SEQ ID NOs: 21-25 are substituted.
  • one or more amino acid is substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the substituted amino acid is at position 3 or position 8. In some embodiments, the substituted amino acid is at position 8 and is substituted with a Cys. In some embodiments, the substituted amino acid is at position 3 and is substituted with a Cys. 42 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Exemplary Cys substituted peptides and peptidomimetic sequences are listed in TABLE 4. In some embodiments, the Cys substituted peptide is selected from the group of SEQ ID NOs: 26- 34. [0154] TABLE 6 lists exemplary amino acid sequences of peptidomimetics disclosed herein.
  • the amino acid sequence of the peptide or peptidomimetic comprises VMAPRTLFL (SEQ ID NO:36) or VMAPRT(L/V)(V/L/I/F)L with one or more amino acid substitutions.
  • the amino acid sequence of the peptide or peptidomimetic comprises wherein one or more amino acids that are substituted with a Cys, Lys, Tyr, His, Ser, or Thr.
  • the amino acid sequence of the peptide or peptidomimetic comprises a substitution of the V residue at a position 1; a substitution of the M residue at a position 2; a substitution of the A residue at a position 3; a substitution of the P residue at a position 4; a substitution of the R residue at a position 5; a substitution of the T residue at a position 6; a substitution of the L residue at a position 7; a substitution of the F residue at a position 8; a substitution of the L residue at a position 9; or a combination of any of the foregoing substitutions.
  • the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino 43 IPTS/128777599.4 Attorney Docket No.: CLS-039WO acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg.
  • the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAR. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr.
  • the synthetic peptides and peptidomimetics are designed to bind HLA-E.
  • the synthetic peptides and peptidomimetics bind HLA-E with low, medium, or high affinity.
  • the synthetic peptides and peptidomimetics bind HLA-E with higher affinity than VL9.
  • the synthetic peptides and peptidomimetics are covalently bound to HLA-E.
  • the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln-72, Asn-77, Thr-80, Tyr-84, Trp-97, His-99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser-147, Glu-152, His-155, Gln- 156, Tyr-159, Thr-163, Cys-164, Trp-167, or Tyr-171 of human HLA-E.
  • the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E.
  • the synthetic peptides and peptidomimetics are listed in TABLE 13, TABLE 14, TABLE 15, TABLE 16, TABLE 17, TABLE 18, or TABLE 19.
  • Peptide Libraries [0159] The inventors of the present disclosure demonstrated the ability to use AS-MS to discover novel peptide binders from libraries with up to 200 million synthetic members in a single experiment 27-29 and enable the identification of peptidomimetic modulators of the interaction between HLA-E and CD94–NKG2A.
  • synthetic peptide libraries can be readily prepared by solid-phase peptide synthesis (SPPS) and allow the facile introduction of non-natural amino acids into a library design to extend the chemical diversity.
  • SPPS solid-phase peptide synthesis
  • the resulting non-natural peptides can displace VL9 from the antigen- binding groove of HLA-E and inhibit its molecular recognition by CD94–NKG2A by design. In some embodiments, this can be validated by fluorescence polarization (FP), liquid chromatography-mass spectrometry (LC-MS), and biolayer interferometry (BLI) assays.
  • FP fluorescence polarization
  • LC-MS liquid chromatography-mass spectrometry
  • BLI biolayer interferometry
  • the peptides or peptidomimetics stabilize the HLA-E/B2M complex, as 44 IPTS/128777599.4 Attorney Docket No.: CLS-039WO determined, e.g., by a thermal shift assay.
  • a synthetic peptide or peptidomimetic as disclosed herein e.g., peptide B9 can stabilize HLA-E on the surface of cells to a similar extent as a comparator peptide (e.g., exogenous VL9, Mtb44, etc.), optionally while leading to a dose-dependent loss of co-receptor engagement in a purified format and/or on reporter cells overexpressing CD94–NKG2A.
  • the peptide library has the design X1- Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu (SEQ ID NO: 3) or X 1 -Met-X 3 -X 4 -X 5 -Ala-X 7 -X 8 -Leu-Lys (SEQ ID NO: 4) is generated, wherein each of X 1 , X 3 , X 4 , and X 7 are independently selected from Gly, Ala, Met, Pro, Cpa, Cha, Ser, Asn, Gln, Msn, Phe, Tyr, His, Trp, 4Py, 4Af, Tha, Dff, Asp, Glu, Lys, Arg, hAr, Aad; X 5 is Gly, Ala, Val, Leu, Met, Pro, Cpa, Cba, Cha, Aoa, Phe, Trp, Mff, Dff, Tff, Tha, Na
  • the half of the library has a Lys at the C- terminus.
  • anchor residues Met2 and Leu9 are set constant in the library design.
  • Arg5 is substituted with aliphatic and aromatic monomers, and Phe8 is replaced by polar and charged residues.
  • 21 non-canonical amino acids are included in the library design.
  • the library is 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million peptides in size.
  • the library is 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, or 100 million, or 200 million peptides in size.
  • Determination of the crystal structure of, e.g., peptide B9 in complex with HLA-E/B2M confirmed the location of peptides (e.g., B9) in the antigen-binding groove of HLA-E with the expected anchor residues.
  • the otherwise linear peptide B9 as disclosed herein, can adopt a characteristic kinked conformation of its backbone presenting functional groups to the solvent-exposed surface intended to inhibit interactions with CD94–NKG2A.
  • each of the peptides disclosed herein are contemplated as lipidated peptides.
  • that lipidation involves the incorporation of a lipid moiety and spacer (e.g. linker) into the disclosed synthetic peptides or peptidomimetics.
  • that incorporation is such that one or more amino acids is substituted with a Lys- R y ; and wherein the R y comprises C18 octadecanedioic acid and, optionally, a linker.
  • that incorporation is such that one or more amino acids is substituted with a Lys-R y ; and wherein the R y comprises a lipid.
  • the R y comprises a hydrocarbon-based compound comprising two -COOH carboxyl functions.
  • the R y comprises a saturated diacid.
  • the R y comprises an unsaturated diacid.
  • R y comprises ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, tetradecanedioic acid (C14 diacid), hexadecanedioic acid (C16 diacid), heptadecanedioic acid (C17 diacid), octadecanedioic acid (C18 diacid), nonadecanedioic acid (C19 diacid), eicosanodioic acid (C20 diacid), heneicosanodioic acid (C21 diacid) myristic acid, docosano
  • Non-limiting examples of unsaturated diacids include (Z)-butenedioic acid (maleic acid), (E)-butenedioic acid (fumaric acid), (Z and E)-pent-2-enedioic acid (glutaconic acid), 2- decenedioic acid, dodec-2-enedioic acid (traumatic acid), (2E,4E)-hexa-2,4-dienedioic acid (muconic acid), and mixtures or combinations thereof [0166]
  • R y comprises a C14 diacid group, C16 diacid groups, or C18 diacid group.
  • R y comprises a lipid moiety means for improving pharmacological properties, e.g. stability and half-life.
  • the incorporation of lipid moieties into peptides can yield improved pharmacological attributes, encompassing heightened stability and subsequent extension of the drug’s in vivo half-life.
  • the present disclosure demonstrates an efficacious method for altering the pharmacokinetic and pharmacodynamic characteristics of primary peptide therapeutics.
  • the inclusion of lipid groups onto a peptide scaffold has the potential to significantly elevate enzymatic stability, receptor specificity, potency, bioavailability, and drug delivery capabilities.
  • lipid moieties such as C18 octadecanedioic acid group attached to Lys-26, in some embodiments, along with an extended spacer, e.g., composed of ⁇ -Glu linked to two 8-amino-3,6-dioxaoctanoic acid groups, improved the pharmacological properties (e.g., stability and half-life) of the peptides.
  • lipidation of a residue in any one of the disclosed peptides can effectively improve the peptides or peptidomimetics pharmacological properties (e.g., stability and half-life).
  • a lipidated peptide or peptidomimetic has enhanced stability and bioactivity, e.g., in serum-containing media, relative to its unlipidated counterpart or a comparator.
  • lipid-modified B9 stabilizes HLA-E at the surface of target cells and leads to dose-dependent cytotoxic cell-killing by human NK cells. III.
  • the disclosure is directed to synthetic peptides and peptidomimetics that are chemically modified.
  • the peptide or peptidomimetic that is modified is selected from TABLE 3, TABLE 4, TABLE 5, or TABLE 6.
  • the peptide or peptidomimetic that is modified is selected from SEQ ID NO: 1-36. Modifications may comprise chemical modifications for example such as warheads, protective groups, and pegylation.
  • the modification is at the N- or C-terminus of the peptide or peptidomimetic. In some embodiments, the modification is on a side chain of an amino acid in 47 IPTS/128777599.4 Attorney Docket No.: CLS-039WO the peptide or peptide. In some embodiments, the modification is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the modification is an arylation. In some embodiments, the arylation is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the modification is acetylation, formylation, propanoylation, hexanoylation, or myristoylation.
  • the modification is an amidated C-terminus. In some embodiments, the modification is a substitution of one or more L-amino acid with a D-amino acid. In some embodiments, the modification is a substitution of one or more amino acid with a methyl-amino acid. In some embodiments, the modification is a substitution of an ⁇ -amino acid with a ⁇ -amino acids. In some embodiments, the arylation is on a Cys, and the Cys is a position 3 or 8 of the synthetic peptide or peptidomimetic. Warheads [0172] In some aspects, the disclosure is directed to synthetic peptides and peptidomimetics comprising a warhead.
  • the peptide or peptidomimetic that is modified with a warhead is selected from TABLE 3, TABLE 4, TABLE 5, or TABLE 6. In some embodiments, the peptide or peptidomimetic that is modified is selected from SEQ ID NO: 1-36.
  • the warhead facilitates a covalent bond to a cognate protein after a chemical reaction. In some embodiments, the warhead is at the N- or C-terminus of the peptide or peptidomimetic. In some embodiments, the warhead is on a side chain of an amino acid in the peptide or peptide. In some embodiments, the warhead is on a Cys, Lys, Tyr, His, Ser, or Thr.
  • the warhead connected to the peptide by an arylation.
  • the arylation is on a Cys, Lys, Tyr, His, Ser, or Thr.
  • the warhead is on a Cys, and the Cys is a position 3 or 8 of the peptide or peptidomimetic.
  • the warhead is conjugated to the Cys via the Sulfur atom of the Cys.
  • the peptide or peptidomimetic with a warhead has the following formula 48 IPTS/128777599.4 Attorney Docket No.: CLS-039WO .
  • the peptide or peptidomimetic with a warhead has the following formula . [0175] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula . [0176] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula . [0177] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula 49 IPTS/128777599.4 Attorney Docket No.: CLS-039WO . [0178] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula .
  • the peptide or peptidomimetic with a warhead has the following formula .
  • the peptide or peptidomimetic with a warhead has the following formula . 50 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0181] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula .
  • the warhead is 52 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein X is a halogen. [0187] In some embodiments, the warhead is . [0188] In some embodiments, the warhead is selected from the group consisting of 53 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0189] In some embodiments, the warhead is selected from the group of a sulfonyl fluoride, a phenyl carbamate, and a squaramate. In some embodiments, the warhead is conjugated to a Cys via the Sulfur atom of the Cys.
  • Cys substituted peptides and peptidomimetic sequences with a warhead are listed in TABLE 7.
  • the Cys substituted peptide with a warhead is selected from the group of SEQ ID NOs: 37-46.
  • TABLE 7 lists exemplary amino acid sequences of peptide and peptidomimetics with a warhead disclosed herein.
  • TABLE 7 54 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0191]
  • the peptide or peptidomimetic with a warhead has the following formula .
  • the peptide or peptidomimetic with a warhead has the following formula .
  • the peptide or peptidomimetic with a warhead has the following formula . 55 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0194] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula . [0195] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula . [0196] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula .
  • the peptide or peptidomimetic with a warhead has the following formula . [0198] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula . [0199] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula 57 IPTS/128777599.4 Attorney Docket No.: CLS-039WO IV.
  • the disclosure is directed to synthetic peptides and peptidomimetics that are bound in a complex with HLA-E/ ⁇ 2m to form peptide/HLA-E/ ⁇ 2m.
  • the peptide or peptidomimetic that is complexed with HLA-E/ ⁇ 2m is selected from TABLE 3, TABLE 4, TABLE 5, TABLE 6, or TABLE 7.
  • the peptide or peptidomimetic that is complexed with HLA-E/ ⁇ 2m is selected from SEQ ID NOs: 1-47.
  • the synthetic peptides and peptidomimetics are designed to bind HLA-E/ ⁇ 2m.
  • the synthetic peptides and peptidomimetics bind HLA- E/ ⁇ 2m with low, medium, or high affinity. In some embodiments, the synthetic peptides and peptidomimetics bind HLA-E with higher affinity than VL9. In some embodiments, the peptides and peptidomimetics are covalently bound to HLA-E/ ⁇ 2m.
  • the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln-72, Asn-77, Thr-80, Tyr-84, Trp-97, His- 99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser-147, Glu-152, His-155, Gln-156, Tyr- 159, Thr-163, Cys-164, Trp-167, or Tyr-171 of human HLA-E.
  • the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E.
  • the peptide/HLA-E/ ⁇ 2m is modulated in binding of CD94/NKG2A.
  • the peptide/HLA-E/ ⁇ 2m is inhibited in binding or engaging of CD94/NKG2A or prevents activation of CD94/NKG2A.
  • the peptide/HLA-E/ ⁇ 2m is located on a cell.
  • the peptide/HLA-E/ ⁇ 2m is soluble.
  • the cell is a cancer cell.
  • the CD94/NKG2A is located on a NK cell or a T cell.
  • the inhibition of binding or engaging of peptide/HLA-E/ ⁇ 2m complex to CD94/NKG2A on a NK cell or a T cell modulates activity of the NK cell or the T cell.
  • peptides can be chemically synthesized using the sequence information provided herein and using peptide synthesis methods known in the art.
  • the produced synthetic peptide or peptidomimetic can be modified during or after peptide synthesis with several modifications, for example with a warhead, a protective group, or pegylation.
  • the peptide or peptidomimetic may be 58 IPTS/128777599.4 Attorney Docket No.: CLS-039WO modified at its amino terminus or carboxy terminus or protected by various organic groups for protecting the peptide from protein-cleaving enzymes in vivo while increasing its stability.
  • the produced synthetic peptide or peptidomimetic can then be purified further.
  • the present disclosure provides an isolated nucleic acid encoding a synthetic peptide or peptidomimetic as disclosed herein or fragment thereof.
  • the present disclosure provides one or more vectors encoding an isolated nucleic acid as disclosed herein.
  • the peptides or peptidomimetics are contemplated as recombinant peptides.
  • the present disclosure provides one or more host cells comprising a vector or isolated nucleic acid as disclosed herein.
  • Non-limiting examples of host cells include Bacterial Cells (e.g., Escherichia coli), Yeast Cells (e.g., Saccharomyces cerevisiae), and Mammalian Cells (e.g., CHO (Chinese Hamster Ovary), HEK293 (Human Embryonic Kidney) cells, etc.).
  • Bacterial Cells e.g., Escherichia coli
  • Yeast Cells e.g., Saccharomyces cerevisiae
  • Mammalian Cells e.g., CHO (Chinese Hamster Ovary), HEK293 (Human Embryonic Kidney) cells, etc.
  • CHO Choinese Hamster Ovary
  • HEK293 Human Embryonic Kidney
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see, e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. 59 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Co., Easton, PA [1975].
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • Pharmaceutical compositions containing a synthetic peptide or peptidomimetic disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration, e.g., oral administration.
  • the pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration.
  • Sterile solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • pharmaceutically acceptable excipient refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • compositions of the invention are any of those that are well known in the art of pharmaceutical formulation and include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils.
  • compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum 60 IPTS/128777599.4 Attorney Docket No.: CLS-039WO albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated
  • the synthetic peptides and peptidomimetics of the disclosure can be used in a variety of in vitro and in vivo methods, as research reagents, for diagnostic purposes, and for therapeutic uses, based on the binding specificity of the synthetic peptides and peptidomimetics to HLA-E and on the effect on HLA-E functions of the peptides and peptidomimetics.
  • the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in a therapy (e.g., cancer).
  • the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in in the preparation of a medicament.
  • a method of treating a disease or condition comprising administering a therapeutically effective amount of a synthetic peptide or peptidomimetic as disclosed herein.
  • the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in the preparation of a medicament for directly and/or indirectly enhancing tumor cell death, the method comprising exposing a tumor (or one or more tumor cells) and natural killer cells to the synthetic peptide or peptidomimetic.
  • the cancer or tumor cells are HLA-E-expressing.
  • Approximately half of peripheral NK cells display the CD94/NKG2A receptor and these cells are mostly present in the CD56high fraction, which contains the more immature cells.
  • Intratumoral NK cells have somewhat higher frequencies of CD94/NKG2A.
  • CD94/NKG2A is also expressed on intratumoral CD8+ T cells that often display a late effector memory phenotype.
  • the inhibitory signals induced by NKG2A receptor engagement with peptide/HLA-E results in decreased capacity of NK cells and CD8+ T cells to lyse target cells.
  • the synthetic peptides and peptidomimetics disclosed herein are designed to be recognized and bound covalently or non-covalently by HLA-E/ ⁇ 2m complexes.
  • the synthetic peptides and peptidomimetics can be used to modulate HLA-E/ ⁇ 2m function.
  • the synthetic peptides and peptidomimetics in complex with HLA-E modulate HLA-E engagement with the CD94/NKG2A receptor heterodimer on NK or T cells. In some embodiments, the synthetic peptides and peptidomimetics in complex with HLA- E block or inhibit HLA-E engagement with the CD94/NKG2A receptor heterodimer on NK or T cells.
  • the HLA-E/ ⁇ 2m complex is presented on the surface of a cancer cell and the peptide or peptidomimetic binds to the HLA-E/ ⁇ 2m complex in a manner that blocks the HLA-E/ ⁇ 2m complex from engaging with the CD94/NKG2A receptor on NK or T cells.
  • Methods for testing for peptide/HLA-E/ ⁇ 2m -CD94/NKG2A engagement and subsequent cell signaling are known in the art, for example by FACS, cytotoxicity assays, and cytokine release assays. IX.
  • kits [0217] In some embodiments, any of the synthetic peptides or peptidomimetics disclosed herein disclosed herein is assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic or research applications.
  • a kit may include one or more containers housing any of the systems or vectors disclosed herein and instructions for use.
  • the kit may be designed to facilitate use of the methods described herein by researchers and can take many forms.
  • Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.
  • EXAMPLE 1 - Peptide Library Design and Synthesis This example describes the design of a peptide library based on the HLA-E signal peptide VL9 VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO:1 ) (VMAPRTLVL, SEQ ID NO:2 ) and subsequent library synthesis of a library of 200 million peptides synthesized by split-and-pool synthesis.
  • HLA-E/VL9/CD94/NKG2A complex crystal structure (the crystal structure is represented in PDB ID 3cii).
  • VL9 binds the groove formed by two ⁇ -helices of HLA-E and several amino acids within VL9 were found useful for anchoring to HLA-E (e.g., Met2 and Leu9, FIG. 1B) and binding to the receptor dimer (e.g., Arg5, Phe8).
  • Arg-5, Thr-6, and Phe-8 of VL9 were shown to be key residues involved in molecular recognition by CD94–NKG2A.
  • the library also introduced orthogonal functional groups to replace interacting residues Arg-5, Thr-6 and Phe-8.
  • C-terminal Lys was installed on half of the library to increase sequencing confidence by augmented signal intensity of fragments in secondary mass spectra.
  • the 21 non-canonical amino acids were included in the library design.
  • SPPS split-and-pool synthesis
  • the 200 million-member library was used in affinity selections with immobilized HLA-E/B2M in complex with a UV-cleavable VL9 to identify putative binders by tandem mass spectrometry and de novo peptide sequencing (FIG. 1E).
  • a peptide library with 200 million members was synthesized on Tentagel® M NH 2 resin (30 ⁇ m bead size, 0.72 mmol, 2.79 g, 70 million beads/g). The resin was placed in a fritted syringe and swollen in N,N-dimethylformamide (DMF) for 30 min.
  • DMF N,N-dimethylformamide
  • HMPB 4 ⁇ (4 ⁇ Hydroxymethyl ⁇ 3 ⁇ methoxyphenoxy)butyric acid
  • DIEA N,N-diisopropylethylamine
  • the reactants were removed by washing with DMF (3x), and the resin was split into two parts to couple the C- terminal amino acids Fmoc-Leu-OH, and Fmoc-Lys(Boc)-OH for the 9-mer and 10-mer library members, respectively.
  • the amino acids (7 equiv.) were dissolved in DMF (0.5 M), N,N’- diisopropylcarbodiimide (DIC, 5 equiv.) was added, and the solution was added to the resin after 2 min of activation.
  • 4-Dimethylaminopyridine (DMAP, 0.1 equiv.) was added, and the reaction was left for 16 h.
  • the Fmoc protecting group was removed with 20% piperidine in DMF (2 x 5 min).
  • Fmoc-Leu-OH (5 equiv.) was coupled to the resin functionalized with Lys with HATU (4.5 equiv) and Dipea (15 equiv) in DMF for 15 min, and, after washing and Fmoc deprotection, the resins were combined for split and pool synthesis was continued by coupling amino acids (7 64 IPTS/128777599.4 Attorney Docket No.: CLS-039WO equiv.) by activation with HATU (6.3 equiv.) and DIEA (21 equiv.) for 15 min at RT followed by Fmoc deprotection according to the library design.
  • EXAMPLE 2 Protein Expression and Purification [0226] This example describes the expression, purification, and refolding of HLA-E and ⁇ 2m protein from inclusion bodies in E. coli and the expression and purification of CD94/NKG2A single-chain dimer from mammalian cells.
  • HLA-E and B2M proteins from inclusion bodies [0227]
  • the coding sequences for HLA-E*0103 (human, residues 22-305)(SEQ ID NO:48) with a C-terminal Avitag and ⁇ 2m (human, residues 21-119) (SEQ ID NO:49) were synthesized and cloned into pET29b(+) (pET29b(+)-HLAE*(hu)(22-305)-Avitag and pET29b(+)-( ⁇ 2m (h)(21- 119)) (synthesized at Genewiz). [0228] The proteins were expressed in E.
  • pellets from 10 L cultures were resuspended in 200 mL of sucrose buffer (50 mM Tris pH 8.0, 1 mM EDTA and 25% sucrose), lysed with the addition of 0.2 g of lysozyme. After 10 minutes of lysis the solution was diluted with 500 mL of deoxycholate solution (20 mM Tris pH 7.5, 100 mM NaCl, 1% deoxycholic acid, and 1% Triton).
  • pellets were resuspended in urea solution (25 mM MES pH 6.0, 8 M urea, 10 mM EDTA, and 0.1 mM DTT). Protein concentrations were determined by A 280 using extinction coefficients for individual proteins. Refolding of biotinylated HLA-E complexes [0229] MHC complexes were refolded as previously described in Braud et al., Nature 1998, 391 (6669), 795-799 and Altman et al., Science 1996, 274 (5284), 94-96.
  • VMAPRTLFL VL9 (SEQ ID NO: 36) to form HLA-E+VL9, VMAP (C/5-Carboxyfluorescein)TLFL (VL9*5FAM), or VMAP(Anpp)TLFL (SEQ ID NO: 47)
  • VMAPRTLFL VL9
  • VMAP C/5-Carboxyfluorescein
  • VL9*5FAM VMAP(Anpp)TLFL
  • VMAP(Anpp)TLFL SEQ ID NO: 47
  • UV- labile VL9, VL9 UV to form HLA-E+VL9 UV by dilution into 1 L of refolding buffer (400 mM L- arginine, 100 mM Tris, pH 8.0, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione).
  • each protein was diluted with 4 mL of injection buffer (3 M guanidine HCl, 10 mM sodium acetate, and 10 mM EDTA).
  • injection buffer 3 M guanidine HCl, 10 mM sodium acetate, and 10 mM EDTA.
  • Refolding solution was then subjected to buffer exchange by tangential flow filtration over a 30K MWCO PALL Omega TFF Cassette into 20 mM Tris pH 8.0.
  • MHC complexes were then purified on an AKTA PURE using a 5 mL HiTrap Q FF column with a 25 CV gradient from 0 to 500 mM NaCl.
  • CD94-NKG2A single-chain dimer expression and purification [0230] A single-chain dimer of CD94/NKG2A (hSCD) was generated by fusing human CD94 (K32-I179, Uniprot Q13241) via a GS(G4S) 7 GG linker to human NKG2A (P94-L333, Uniprot P26715) (SEQ ID NO:50). An N-terminal 8xHis tag was added for IMAC purification, and the whole construct was subcloned into a mammalian expression vector with a puromycin selection cassette.
  • aFc-hSCD (SEQ ID NO:51)
  • the N-terminal 8xHis tag was replaced by an AviTagTM followed by a one-armed human IgG1 Fc (L234A, L235A, L351K, T366S, P395V, F405R, Y407A, K409Y), linked to the hSCD insert with a GGG linker.
  • Stable cell lines for both constructs were generated in HEK293F cells using puromycin selection.
  • HBS 50 mM HEPES, 300 mM NaCl, pH 7.5
  • HBS 50 mM HEPES, 300 mM NaCl, pH 7.5
  • the protein was eluted using a gradient elution of 20 mM – 1 M imidazole in HBS over 6 CV.
  • Fractions were pooled, concentrated, and passed through a Superdex® 200 Increase 10/300 GL column (Sigma GE28-9909-44) in HBS.
  • the filtered conditioned media was loaded on a 5 mL HiTrapTM Protein G HP (Cytiva, 17-0404-01) on an Akta FPLC at 1mL/min flow rate.
  • the column was then washed with 10 CV of PBS (Corning 21-040-CV).
  • the protein was eluted using 0.1 M acetic acid (pH 2.7) and immediately neutralized with 1:10 the volume of 1 M Tris pH 8 and 1 M NaCl. Peak fractions were pooled, concentrated, and injected onto a Superdex® 200 Increase 10/300 GL column (Sigma GE28- 9909-44) equilibrated in PBS running at 0.5 mL/min. The main peak was collected and further concentrated for downstream applications. The final protein concentration was quantified using PierceTM 660 nm Protein Assay Reagent (Thermo 22660) before the protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C until use.
  • EXAMPLE 3 Peptide Selection by Binding to HLA-E [0231] This example describes the selection of HLA-E peptide binders from the focused library by nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS). Since the HLA- E/B2M complex requires a peptide ligand for stability, the biotinylated HLA-E/B2M complex was first charged with a UV-cleavable analog of VL937 (abbreviated, e.g., as HLA- E/B2M/VL9 UV ).
  • VL937 abbreviated, e.g., as HLA- E/B2M/VL9 UV
  • the beads were subjected to three cycles of suspending in 1 mL wash buffer followed by separation enabled by a magnetic rack.
  • the washed beads were suspended in PBS (+ 10% FCS), and the library was added a concentration of 10 pM/member in 1.7 mL centrifuge tubes. Selections were performed under UV irradiation to cleave UV-labile VL9 and liberate the binding groove of HLA-E in presence of the peptide library.
  • the magnetic beads bearing the binder–target complexes were removed from the mixture and washed to remove non-binding sequences. After incubation, the solution was removed on the separating rack, and the beads were subjected to three cycles of wash with PBS and separation enabled by the magnetic rack. Finally, the beads were treated with 6 M guanidine in 0.2 M phosphate buffer (pH 6.8) to denaturate the proteins and elute bound peptides.
  • the samples were desalted by a C18 ZipTip prior to lyophilization, and dissolved in 100 mM guanidine in H 2 O (+ 0.1% formic acid) for analysis by nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS) on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer.
  • LC was performed with 0.1% formic acid (FA) in water (solvent A) and 80% MeCN with 0.1% formic acid in water (solvent B) prepared with LiChrosolv® water and MeCN suitable for MS from Millipore Sigma and Optima TM LC/MS grade formic acid from Thermo Fisher Scientific. Chromatography was performed at 40 ° ⁇ , with a flow rate of 300 nL/min using either of the following gradient: 1% B to 45% B (0–100 min), 45% B to 90% B (100–102 min), 90% B (102–100 min) or 1% B to 51% B (0–120 min), 51% B to 90% B (120–130 min), 90% B (130–140 min).
  • EXAMPLE 4A Inhibition of CD94–NKG2A Binding to HLA-E by Peptides
  • This example describes the analysis of inhibition of CD94–NKG2A binding to HLA-E by the peptides B1–B5 by bio-layer interferometry (BLI).
  • BKI bio-layer interferometry
  • B5 derived amino acid sequences [0243] Briefly, peptides were synthesized by SPPS on a 0.05 mmol scale with a HMPB ChemMatrix resin in fritted syringes. The C-terminal amino acid (7 equiv.) was coupled to the resin by DIC (5 equiv.) and DMAP (0.1 equiv.) for 16 h at rt.
  • the Fmoc protecting group was 71 IPTS/128777599.4 Attorney Docket No.: CLS-039WO removed using 20% piperidine in DMF for 2 x 5 min, and the subsequent amino acids (5 equiv.) were coupled using HATU (4.5 equiv.) and DIEA (15 equiv.) in DMF for 15 min at room temperature (rt).
  • the resin was washed with DMF and CH 2 Cl 2 , and dried in a vacuum chamber for 16 h.
  • Ligand exchange of the peptides B1–B5 with VL9 on HLA-E was studied by incubating [HLA-E+VL9] with the competitor peptides. [0246] Briefly, 10 ⁇ M competitive peptides, VL9 peptide, or DMSO were added to 1 ⁇ M of refolded HLA-E/B2M complexes loaded with VL9 in 1X PBS (Corning 21-040-CV) and allowed to exchange for 4 h at 25 °C.
  • HLA-E Peptide Exchange Analysis by SEC and LC-MS [0247] To determine the exchanged peptides in the HLA-E/B2M complexes, the exchanged complexes were analyzed by SEC and LC-MS. Briefly, an Agilent 1200 Series Infinity II HPLC coupled to an analytical fraction collector (Agilent, G1364F) was used to separate peptide- loaded HLA-E/B2M protein from free peptide after peptide exchange as described above. Briefly, 50 ⁇ L of peptide-exchange solution was loaded onto a Superdex 200 Increase size exclusion column (Cytiva, 28990945) at a flow rate of 0.45 mL/min in PBS at room temperature.
  • the main UV absorbance peak at 280 nm was collected over a 200 ⁇ L volume fraction for downstream analysis. 20 ⁇ L of the collected fraction was injected into an Agilent 1200 Series Infinity II HPLC with a 2.1 x 50mm ZORBAX 80 ⁇ Extend-C18 reverse phase column (Agilent, Part Number: 727700-902) equilibrated in water with 0.1% (v/v) formic acid and 10% (v/v) acetonitrile flowing at 0.2 mL/min with a column temperature of 40 °C.
  • peptide peaks were resolved using a 10 – 60 % gradient of acetonitrile in 0.1% (v/v) formic acid and eluted into a Dual Agilent Jet Steam electrospray ionization source operating at a Gas Temp of 350 °C, Drying Gas at 10 L/min, Nebulizer at 30 psig, Sheath Gas Temp at 350 °C, Sheath Gas Flow at 11 L/min, VCap voltage at 3500 V, and Nozzle Voltage at 1000 V.
  • Peptide ions were detected with an Agilent 6230 Time-of-Flight mass spectrometer operating in positive ion mode with a Fragmentor Voltage of 150 V and Skimmer Voltage of 65 V.
  • HLA-E/B2M/VL9 VMAPRTLFL
  • Biotinylated [HLA-E+VL9] (1 ⁇ M) was co-incubated with individual peptides B1- B5 (10 ⁇ M) overnight at room temperature, and the biotinylated complex was subsequently loaded on streptavidin-coated probes for analysis by BLI. (FIG. 14B).
  • Binding inhibition of CD94–NKG2A (200 nM) to the HLA-E-loaded probes was determined relative to the parent [HLA-E+VL9] complex.
  • Experimental controls with VL9 or DMSO during ligand exchange were included to compare binding of CD94–NKG2A to their cognate ligand.
  • Peptides B2 and B3 showed partial inhibition of CD94–NKG2A binding to HLA-E, whereas almost no residual binding was observed for peptide B5.
  • Peptides B1 and B4 displayed no inhibitory effect in the BLI assay (FIG. 14C). [0250] BLI was performed on a ForteBio Octet Red96e instrument.
  • HLA-E+VL9 biotinylated [HLA-E+VL9] complexes were loaded onto SA biosensors (Forte 18- 5019) at ⁇ 5 ug/ml (100nM) with the following steps: 60 sec baseline in 1X Kinetic Buffer, 180 sec loading, 60 sec blocking in Blocking Buffer and 60 sec baseline in 1X Kinetic Buffer, all at 25 °C and 1000 rpm.
  • hSCD and oaFC-hSCD binding to immobilized HLA-E/B2M complexes was monitored at 200 nM by a 90 sec association step, followed by a 120 sec dissociation step.
  • B2, B3, and B5 showed 41–98%. inhibition of CD94/NKG2A binding in the biophysical assay. In particular, B5 showed only 2% of residual binding of CD94/NKG2A compared to untreated control (FIGs. 5C-5E).
  • a series of peptides with single substitution of anchor residues Met2 or Leu9 of B5 (B9, B10, B6, B7, and B8) showed comparable inhibition of CD94/NKG2A binding (FIGs.
  • peptide-stabilized HLA-E/B2M complexes were isolated after ligand exchange using size- exclusion chromatography and subsequently subjected to analysis by LC-MS to quantify the remaining fraction of VL9 in the fully-folded complexes (FIG. 14D).
  • B1 only displaced minimal amounts of VL9 from the HLA-E groove while B4 did not (FIG. 14E).
  • Octet® High Precision Streptavidin (SAX) Biosensors [Sartorius 18-5117 ] were allowed to equilibrate in 1X KB for at least 10 min at room temperature while the plate was prepared.
  • Blocking buffer was prepared by adding 5% BSA [Sigma A3294] and 20 ug/mL Biocytin [Sigma B4261] to 1X KB and used to quench any remaining binding sites on the SAX tips before association with the SCD. Tips were rinsed in 1X KB, HLA-E complex adsorbed onto the tips, blocked with blocking buffer, rinsed with 1X KB and then allowed to associate with SCD and then rinsed in 1X Kinetic Buffer to allow for dissociation of the SCD.
  • the plate was allowed to shake at 1000 rpm at room temperature for the duration of the assay.
  • the steps of the assay are as follows: Baseline 60s, Loading 180s, Blocking 60s, Baseline 60s, Association 180s, Dissociation 240s.
  • Raw traces were observed for loading of complex and then traces were aligned to Y axis to the average of the Baseline step.
  • the inter-step correction 75 IPTS/128777599.4 Attorney Docket No.: CLS-039WO aligned the data to the Dissociation step, and then Savitzky-Golay filtering was used to remove high-frequency noise.
  • Data was analyzed on the Data Analysis HT software [Sartorius] and exported for Prism [Graphpad].
  • Fluorescence polarization (FP) assay [0255] To further understand the kinetics involved in ligand exchange, a fluorescence polarization (FP) assay was developed based on purified HLA-E/B2M complexes bound to a fluorescent analog of VL9 bearing a fluorescein at position 5 (i.e., VL9*5FAM). Hit peptides B1–B5 were added at a ten-fold molar excess, and ligand exchange was monitored at ambient temperature by a loss of FP as the VL9*FAM ligand was released from the complex (FIG. 14F).
  • Peptides were diluted in sterile water and added to the HLA- E/VL9*5FAM/B2M complexes at 1:20 the volume of the reaction.
  • the reaction was read in kinetic mode on a Clariostar plate reader (Ex: 482-16; Dichroic: LP504; Em: 530-40) with shaking at 300 rpm before each reading and 10 min time points over 4 hours.
  • a DMSO control was used to adjust the gains of channel A and B and the mP set to 200.
  • the loss of mP was 76 IPTS/128777599.4 Attorney Docket No.: CLS-039WO measured over approximately 4 h at room temperature and 37°C. Data was analyzed in Prism and normalized by baseline subtraction of the first time point.
  • HLA-E/B2M/VL9 was diluted in 1X PBS (with additive when indicated) and then added to a black-walled 384-well plate (Corning 3575), avoiding the edges of the plate and empty wells filled with 1X PBS to compensate for evaporation. Peptides were diluted in sterile water and added to the HLA-E/B2M/VL9 complexes at 1:20 of the volume of the reaction.
  • the reaction was read in kinetic mode on a Clariostar plate reader (Ex: 482-16; Dichroic: LP504; Em: 530- 40) with shaking at 300 rpm before each reading and 10 min time points over 4 hours.
  • a DMSO control was used to adjust the gains of channel A and B and the mP set to 200.
  • the loss of mP was measured over approximately 4 h at room temperature and 37°C. Data was analyzed in Prism and normalized by baseline subtraction of the first time point.
  • HLA-E/VL9 complex after incubation with selected peptides for exchange was injected through an Agilent Infinity II HPLC system onto a Superdex 200 Increase 5/150GL size exclusion chromatography column (Cytiva 28990945) and fractions of the HLA-E/Peptide complex were collected to isolate peptides bound onto HLA-E away from free unbound peptides.
  • the HLA-E/Peptide complex was then injected onto another Agilent Infinity II HPLC system onto a MabPac RP, 4uM, 2.1X50mm reverse phase column (ThermoFisher 088648) in tandem with an Agilent 6230 Time-Of-Flight mass spectrometer equipped with Dual Agilent Jet Stream electrospray ionization source operating in positive ion mode.
  • Peptides B6, B7, and B8 were synthesized as analogs of peptide B5 featuring non-canonical norleucine (Nle), 8- aminooctanoic acid (Aoa), and ß-cyclohexyl alanine (Cha), respectively, to explore alternative hydrophobic residues at anchor position 9 (FIG. 5A).
  • Nle non-canonical norleucine
  • Aoa 8- aminooctanoic acid
  • Choa ß-cyclohexyl alanine
  • Covalent inhibition is a useful strategy to increase potency, selectivity, and pharmacodynamics of drugs, and alleviates the effects of fast renal elimination of peptides.
  • Several residues in the proximity of VL9 in the binding groove of HLA-E, e.g., Tyr-7, Lys-146, 78 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Tyr-159, Tyr-171, bear nucleophilic groups potentially amenable for covalent binding through a Sulfur(VI) Fluoride Exchange (SuFEx) electrophile contained by the meta-substituted aryl sulfonyl fluoride (mSF) warhead (FIG. 6A).
  • Electrophilic analogs of VL9 were subjected to cross-linking reactions in a 10-fold excess with [HLA-E+VL9] for 4 h at 37 °C, and the amount of covalently bound HLA-E was determined by LC-MS as described in EXAMPLE 4. Installing the electrophilic warhead at positions 1, 2, 5, 6, or 9 led to no or only trace amounts of cross-linking with HLA-E, whereas substantial cross-linking was observed with the aryl sulfonyl fluoride at positions 4 (36%), 7 (22%), and in particular at positions 3 (73%) and 8 (87%) (FIG. 6C). [0269] The electrophilic designer peptide B9_8* (FIG.
  • B2M which is co-expressed with HLA-E for stabilization of the MHC class I molecule and is present at equimolar concentrations in the cross-linking reaction, bears several nucleophilic residues (Lys, Cys, His, Ser, Thr, Tyr). No mass shift was observed for B2M in the cross-linking experiment, indicating that B9_8* binds and reacts specifically with HLA-E (FIG. 8C).
  • Cross- linking reactions with HLA-A, another MHC class I molecule showed no conversion with B9_8* within 4 h.
  • EXAMPLE 6 Determination of Inhibition of CD94–NKG2A Binding to HLA-E by Peptides
  • This example describes the analysis of inhibition of CD94–NKG2A binding to HLA-E by exemplary modified peptides by bio-layer interferometry (BLI).
  • BBI bio-layer interferometry
  • 81 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
  • Additional VL9 based HLA-E binding peptides were designed either based on the library described in EXAMPLE 1.
  • peptides based on the ligand for the NKG2A/CD94 inhibitory receptor in mice the nonclassical MHC molecule Qa-1b, the mouse HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier).
  • Qdm for Qa-1 determinant modifier.
  • This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci.
  • proteins and VL9 or Qdm derived peptides were produced as described in EXAMPLE 2 and EXAMPLE 3. BLI measurements to determine inhibition were performed as described in EXAMPLE 4.
  • TABLE 13 Exemplary modified peptides that were used in the inhibition of CD94–NKG2A binding to HLA-E measured by measurements are shown in TABLES 13, 14, and 15.
  • TABLE 14 shows exemplary Qdm derived peptides and BLI results.
  • TABLE 15 shows exemplary VL9 derived peptides with additional amino acids at the N- terminus and BLI results.
  • TABLE 15 shows exemplary VL9 derived peptides.
  • TABLE 16 84 IPTS/128777599.4 Attorney Docket No.: CLS-039WO EXAMPLE 7 – Determination of Covalent Binding of VL9 modified Analogs to HLA-E [0278]
  • This example describes the analysis of crosslinking of modified VL9 based peptides to HLA-E. [0279] Briefly, crosslinking reactions were performed as described in EXAMPLE 5. Exemplary peptides and crosslinking results are shown in TABLE 17.
  • proteins and VL9 or Qdm derived peptides are produced as described in EXAMPLE 2 and EXAMPLE 3 and binding to HLA-E is measured with Fluorescence Polarization, BLI, or crosslinking as described in EXAMPLE 4, 5, or 7.
  • Exemplary peptides that can be analyzed are shown in TABLE 19.
  • thermostability of HLA-E/B2M complexes was determined after VL9 was exchanged for with Ala-substituted analogs of B9 for 16 h at ambient temperature.
  • substitution of key anchor positions Nle-2 and Leu-9 with alanine revealed the least thermally stable complexes with a melting temperature shift of approximately 6°C below the fully exchanged HLA-E/B2M/B9 complex (FIG. 5J).
  • EXAMPLE 11 Crystal structure of HLA-E bound with peptide B9 [0288]
  • B9 was crystallized in complex with HLA-E and B2M to solve the structure to 1.51 ⁇ resolution (FIG. 15A). All residues of B9 were well resolved with the exception of Mff-5 that featured incomplete sidechain density, possibly due to high flexibility.
  • B9 displayed a similar mode of binding to HLA-E as VL9 at anchor positions 2 and 9 with comparable backbone interactions at position 1 forming a hydrogen bond with the hydroxyl groups of Tyr-7 and Tyr-171 on HLA-E (FIGs.
  • Nle-2 binds in a pocket where it partakes in hydrophobic interactions with Met-45 of HLA-E in a similar manner as observed for Met-2 of VL9 (FIGs. 15C and 15D).
  • HLA-E residue Trp-167 on the ⁇ 2-helix, and Arg-62 and Gln-64 on the ⁇ 1-helix form serial interactions with the side chains of hAr-1 and Gln-4 of peptide B9. Therein, Gln-4 and Mff-5 are covered by Arg-65 of HLA-E (FIG. 15C).
  • the side chain of hAr-3 forms a salt bridge to Glu-114 on the ⁇ -sheet floor and a hydrogen bond to Gln-156 the ⁇ 2-helix in HLA-E and an additional hydrogen bond to the carbonyl oxygen of 97 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Mff-5 in B9 (FIG. 15C).
  • Ala-6 in B9 is located in a pocket formed by hydrophobic interactions with Thr-70 and Ile-73 on the ⁇ 1-helix of HLA-E (FIG.15C).
  • the side chain of residue Cha-7 in B9 forms hydrophobic interactions with Trp-133 and Leu-124 on the ⁇ -sheet floor of HLA-E (FIG. 15C).
  • the sidechain of the anchor residue Leu-9 sits in an adjacent pocket formed by Tyr- 123, Leu-124, and Tyr-118 on the ⁇ -sheet floor of HLA-E (FIG. 15C).
  • the backbone of Leu-9 in B9 forms hydrogen bonds to Asn-77 and Tyr-84 on the ⁇ 1-helix and Ser-143 on the ⁇ -sheet floor of HLA-E, in analogy to VL9, and engages in an additional hydrogen bond with Lys-146 (FIGs. 15C and 15D).
  • Mtb44 contains an arginine at the first position in a similar orientation as hArg-1 in B9, but the extended interaction network as observed with B9 residues Gln-4 and Mff-5 is not present for the 98 IPTS/128777599.4 Attorney Docket No.: CLS-039WO pathogenic peptide because it features residues with short sidechains, Ala-4 and Lys-5 respectively, at these positions (FIG. 15B).
  • Anchor residues at position 2 (Leu in Mtb44 and Nle in B9) assume a similar orientation, and Leu-9 is featured in both peptides in the same binding position, whereas the residues in the center of the peptides differ considerably (FIG. 15B).
  • HLA-E-01:03 protein containing amino acids 22-297 was expressed, purified, and refolded with B2M and B9 peptide as described above. The complex was dialyzed into 20 mM Tris pH 8.0, 100 mM NaCl and concentrated to 13.52 mg/mL for use in crystallization.
  • HLA- E/B2M/B9 complex crystals were grown by hanging drop vapor diffusion with a well solution consisting of 100 mM sodium HEPES/MOPS pH 7.5, 20% PEG 500 MME, 10% PEG 20,000, 30 mM sodium nitrate, 30 mM sodium phosphate dibasic, and 30 mM ammonium sulfate at 18 °C. Crystals were cryo-protected in well solution supplemented with 15% trehalose and flash frozen in liquid nitrogen for data collection. [0292] Data was collected on beamline 8.3.1 at the Advanced Light Source. Data was collected on a single crystal rotated 360° in increments of 0.2° for a total 1200 frames.
  • HLA- E/B2M/VL9 peptide complex (pdb 1ktl) was modified to remove the peptide and used as a search model in Phaser (McCoy et al. 2007) to solve the structure (pdb 1ktl). Phaser fit one copy of the HLA-E/B2M complex into the asymmetric unit of the crystal. Clear electron density for B9 was observed and the peptide was built into it.
  • the structure was manually adjusted to the electron density to add and delete regions that differed from the search model and refined with Phenix (Liebschner et al. 2019).
  • the final structure is composed of HLA-E residues 2-146 and 153-276, B2M residues 1-100 (using the mature protein numbering after signal peptide removal), all B9 residues, one sulfate and four polyethylene glycol molecules (from the crystallization condition), one trehalose molecule (from the cryoprotectant), and 420 waters.
  • EXAMPLE 12A Biophysical Assay of B9 and Mtb44
  • Mtb44 binds to the antigen-binding groove of HLA-E with similar efficiency as VL9, and has been shown to stimulate restricted CD8+ T-cell responses in infected and vaccinated humans.
  • the high stability of Mtb44 in complex with HLA-E/B2M has been extensively 99 IPTS/128777599.4 Attorney Docket No.: CLS-039WO evaluated.
  • this example Given the conformational similarities adopted by B9 and Mtb44 in the peptide- binding groove of HLA-E (FIG. 15B), this example compared these two peptides in biophysical and cellular assays.
  • Control VL9, B9, and Mtb44 and scrambled analogs VL9scr, B9scr, and Mtb44scr were synthesized and analyzed. Both Mtb44 and B9 efficiently displaced VL9 and VL9*5FAM from HLA-E/B2M as analyzed by LC-MS and fluorescence polarization assays (FIGs. 16A and 16B), while the scrambled analog of B9 did not. Given the redundancy in the sequence of Mtb44 (i.e.
  • FIG. 16G a gain of FP assay was designed (FIG. 16G) in which HLA-E/B2M/VL9 complexes were incubated with a 10-fold molar excess of fluorescent analogs of the peptides tagged with fluorescein at position five (i.e., VL9*5FAM, B9*FAM, and Mtb44*FAM, and scrambled analogs). Increases in FP indicative of loading of the fluorescent peptide onto the antigen-binding groove of HLA-E was monitored.
  • EXAMPLE 12B Cellular Assays of B9 and Mtb44
  • B9 is a strong binder to HLA-E/B2M, capable of displacing VL9 and stabilizing HLA-E/B2M in solution
  • this example tested whether it can likewise do so in the context of an HLA-E/B2M complex expressed at the cell surface, and if so, whether it still can efficiently inhibit interactions with soluble and cell-surface expressed CD94–NKG2A co- receptors.
  • First tested was if B9 can efficiently displace VL9 on HLA-E/B2M complexes at the cell surface.
  • Cells were routinely cultured in DMEM [Corning 10-017-CV] + 10% FBS [Gibco] that has been heat-inactivated for 30 min at 56C in Corning® CellBIND® 75cm 2 flasks [Corning 3293] at 37°C (5% CO 2 , 20% O 2 , humidity controlled). Media was sterile filtered before use. To prepare for the assay, cells were plated at 0.1e6 cells per well in a Falcon® 12-well plate [Corning 353043] with IFN- ⁇ [Stemcell Technologies # 78020] stimulation (20 ng/mL) overnight.
  • VL9 was diluted to 1mM in cell culture-grade water [Corning 25- 055-CM] and then added to serum-free DMEM at a final concentration of 20 ⁇ M. Cells were washed twice with 1X PBS and then VL9-media was added to cells. The plate was incubated at 37C for 1 hr. Candidate peptides and their controls were diluted to 1mM in tissue-grade water and then diluted to 20 ⁇ M in serum-free DMEM. VL9 was spiked into the media containing candidate peptides and controls at a final concentration of 2.5 ⁇ M to allow for competition and high surface HLA-E levels.
  • Blocking buffer was prepared by adding BD PharmingenTM Human BD Fc BlockTM (BD Biosciences 564219) to Pharmingen Stain Buffer at a 1:20 v/v dilution. The supernatant was aspirated and 100uL of Blocking buffer added to the wells and the 103 IPTS/128777599.4 Attorney Docket No.: CLS-039WO cells gently resuspended. The tubes were incubated on ice for 30 min.
  • Staining buffers were made while the cells block by diluting APC anti-human HLA-E Antibody (Clone 3D12; Biolegend 342606) to 1:20 in Pharmingen Stain Buffer.
  • Biotinylated Human NKG2A&CD94 Protein, Fc, AvitagTM (Acro Biosystems NC4-H82F5) was diluted to 200nM in Pharmingen Stain Buffer and then Streptavidin, Alexa FluorTM 647 Conjugate (Invitrogen S21374) added at 50nM to allow for tetramerization of the complex before staining.
  • a mouse isotype control, APC Mouse IgG1, ⁇ Isotype Ctrl (FC) Antibody (Clone MOPC-21; BioLegend 400122), and Streptavidin AF647-only control were also included at respective concentrations. After blocking, cells were spun down at 1300 rpm for 5 min at 4C and the supernatant aspirated. Stains were added individually to the tubes and the cells gently resuspended. Cells were incubated for 1 hr at 4°C for 1 hr. After staining, cells were spun down and washed twice with Pharmingen Stain Buffer at the same speed and temperature as above.
  • Jurkat Reporter Assay SCaBER NTC (Venus YFP) were plated in a Falcon® 96-well plate (Corning 353072) with IFN- ⁇ (20 ng/mL) in DMEM + 10% FBS overnight at 37°C. IFN-y was persistent throughout at a concentration of 20 ng/mL. VL9 peptide was diluted to 1mM in cell culture grade water and then diluted in serum-free DMEM to a final concentration of 20 ⁇ M. The plate was washed twice with 1X PBS and the VL9 media was added and the plate incubated at 37°C for 1 hr.
  • Inhibitory peptides were diluted to 1mM in cell culture grade water and then added to serum-free DMEM (containing 10 ⁇ M VL9) to a final concentration of 50 ⁇ M. Peptides were serial diluted 1:2. VL9 was diluted to 20 ⁇ M in serum-free DMEM and serial diluted down to generate a standard curve for Jurkat Reporter interaction. Plates were incubated at 37°C for 2 hr.
  • Jurkat Reporter cells Jurkat E6.1 cells harboring a CAR expression cassette consisting of CD3z-CD28-NKG2A-(ITIM-mut)-P2A-CD94 linked to an NFAT-Lucia secreted luciferase
  • RPMI1640 Cell Culture MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERIAL MATERM /mL
  • FBS heat- inactivated
  • Jurkat Reporter cells were spun down at 1300 rpm, washed once with 1X PBS, and then resuspended in serum-free DMEM to a concentration of 15e 6 /mL. After the 2 hr exchange, 20 ⁇ L of the Jurkat Reporter cells were added to each well to a final concentration of 300K/well.
  • lipidated B9 peptide candidate is also referred to as “B95K lipid”.
  • a control peptide with a scrambled sequence was prepared analogously.
  • Stability Assays 105 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0004] Stability assays were conducted and showed improved serum stability for the lipidated B9 peptide, as shown in FIG. 11. B9 and lipidated B9 were incubated in 25% human serum or 10% FBS for 24 hours and monitored by LC-MS at regular intervals in the presence of a reference peptide (heavy B9). Heavy B9 contains a heavy (isotope) version of Glutamine (Gln, 13C5/15N2, +7 ) purchased from Cambridge Isotope Laboratories, Catalog No. CNLM-7252-H-PK.

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Abstract

This disclosure relates to lipidated synthetic peptides, peptidomimetics, and complexes of synthetic peptides and peptidomimetics with HLA-E, methods of making such peptides, peptidomimetics, and complexes, and methods of using such peptides, peptidomimetics and complexes for blocking, inhibiting, or preventing the interaction of HLA-E with CD94/NKG2A or activation of CD94/NKG2A by HLA-E. The lipidated synthetic peptides, peptidomimetics, and complexes of synthetic peptides and peptidomimetics with HLA-E can further comprise warheads to introduce covalent linkages between the synthetic peptides and peptidomimetics with HLA-E.

Description

Attorney Docket No.: CLS-039WO COMPOSITIONS AND METHODS FOR LIPIDATED PEPTIDE-BASED MODULATORS OF HLA-E CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/602,362, filed on November 22, 2023, the disclosure of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING [0002] This application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on XXX , is named XXX, and is XXX bytes in size. BACKGROUND [0003] Natural Killer cells (NK cells) and T cells play an important role in the innate and adaptive immune response and in the prevention of cancer. These cells provide an efficient immunosurveillance mechanism by which undesired cells such as tumor cells or virally infected cells can be eliminated. NK cell and T cell activity is regulated by a complex mechanism that involves both activating and inhibitory signals. The inhibitory NK cell receptor dimer CD94/NKG2A C-type lectin receptor complex has recently been identified as an immune checkpoint in the tumor microenvironment and is expressed on NK cells as well as some T cell subsets. Interactions between CD94/NKG2A and a peptide- (e.g., VL9-)loaded human leukocyte antigen E (HLA-E) prevent NK cells or T cells from killing healthy cells (FIG. 1A). The expression of HLA-E has also been associated with different types of cancer as a mechanism to evade attacks by NK cells or T cells. High levels of HLA-E expression at the tumor cell surface are reported in several cancer types, including gynecologic cancers (up to 90% of tumor samples, e.g., cervical and ovarian) and up to 50% in breast cancer, non–small cell lung carcinoma (NSCLC), melanoma, glioblastoma, liver, pancreas, kidney, melanoma, prostate, head and neck, stomach, rectal, and colorectal cancer. Blocking of the CD94/NKG2A receptor on NK and T cells has been shown to increase cytotoxic activity of NK and T cells. The overexpression of HLA-E by cancer cells is a potential strategy to escape immune surveillance and protect cancer cells from NK cell-mediated cytolytic attacks. Recently, the antibody Monalizumab, a humanized, CD94-NKG2A-neutralizing, anti-NKG2A antibody, has been shown to enhance NK cell activity against various tumor cells in mouse models and rescued CD8+ T cell function in combination with PD-x axis blockade. 1 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0004] Despite the efforts that have been made to date to block the inhibitory activity of CD94/NKG2A, there is an ongoing need for new and effective treatment modalities for inhibiting CD94/NKG2A function in cancer. Furthermore, there is a need for treatment modalities that have improved pharmacological attributes, such as improved stability and half- life. SUMMARY [0005] The present disclosure is directed, at least in part, to synthetic peptides, peptidomimetics, compositions, and methods for the modulation of HLA-E-CD94/NKG2A interaction (e.g., activation of CD94/NKG2A signaling). [0006] The present disclosure provides a synthetic peptide comprising an amino acid sequence X1-Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3), wherein each of X1, X3, X4, and X7 are independently selected from 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg; X5 is Ala, Cha, Tha, or Mff; and X8 is Arg, Msn, or hAr; wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0007] In some embodiments of Lys-Ry, the linker connects the C18 octadecanedioic acid to the Lys residue. [0008] In some embodiments, the amino acid sequence is (a) NH2-4Af-Met-Dff-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 5); (b) NH2-hAr-Met-Msn-Dff-Cha-Ala-Arg-Msn-Leu-OH (SEQ ID NO: 6); (c) NH2-Ala-Met-hAr-Dff-Tha-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 7); (d) NH2-4Af-Met-Ala-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 8); or (e) NH2-hAr-Met-Har-Gln-Mff-Ala-Cha-hAr-Leu-OH (SEQ ID NO: 9). [0009] In some embodiments, one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 and is substituted with a Cys. In some embodiments, the one or more Cys, Lys, Tyr, His, Ser, or Thr is arylated. In some embodiments, the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead. In some embodiments, the one or more other amino acids is substituted with a Lys and is conjugated to a warhead, wherein conjugation is to the N atom on the side chain. In some embodiments, the one or more other amino acids is 2 IPTS/128777599.4 Attorney Docket No.: CLS-039WO substituted with a Tyr, Ser, or Thr and is conjugated to a warhead, wherein conjugation is to the O atom on the side chain. In some embodiments, the one or more other amino acids is substituted with a His and is conjugated to a warhead, wherein conjugation is to the N atom on the side chain. [0010] In some embodiments, the warhead is
Figure imgf000005_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, - ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, - C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, - C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, - SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0011] In some embodiments, the warhead is
Figure imgf000005_0002
, wherein R2 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, 3 IPTS/128777599.4 Attorney Docket No.: CLS-039WO -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0012] In some embodiments, the warhead is
Figure imgf000006_0001
, wherein R3 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0013] In some embodiments, the warhead is
Figure imgf000006_0002
wherein R10 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - 4 IPTS/128777599.4 Attorney Docket No.: CLS-039WO OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0014] In some embodiments, the warhead is
Figure imgf000007_0001
, wherein X is a halogen. [0015] In some embodiments, the warhead is
Figure imgf000007_0002
. [0016] In some embodiments, the warhead is selected from the group consisting of
Figure imgf000007_0003
5 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000008_0001
[0017] In some embodiments, the warhead is selected from the group of a sulfonyl fluoride, a phenyl carbamate, and a squaramate. [0018] In some embodiments, the warhead is conjugated to the Cys via the sulfur atom of the Cys. [0019] The present disclosure also provides a synthetic peptide comprising an amino acid sequence hAr-X2-hAr-Gln-Mff-A-Cha-hAr-X9 (SEQ ID NO: 20) wherein X2 is Nle or Mox; and X9 is Leu, Aoa, or Cha; wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. In some embodiments, the amino acid sequence is “ (a) NH2-hAr-Nle-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (“B9”; SEQ ID NO: 21); (b) NH2-hAr-Mox-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (“B10”; SEQ ID NO: 22); (c) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Nle-OH (“B6”; SEQ ID NO: 23); (d) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Aoa-OH (“B7”; SEQ ID NO: 24); or (e) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Cha-OH (“B8”; SEQ ID NO: 25). [0020] In some embodiments, the amino acid sequence is NH2-hAr-Nle-hAr-Gln-Mff-Ala-Cha- hAr-Leu-OH (SEQ ID NO: 21). [0021] In some embodiments, the Lys-Ry substitution is at position 5. 6 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0022] In some embodiments, one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys. [0023] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000009_0001
wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0024] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000009_0002
, wherein the R is 7 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000010_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, - ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, - C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, - C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, - SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl; and wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0025] In yet another aspect, the present disclosure provides a synthetic peptide comprising an amino acid sequence VMAPRT(L/V)(V/L/I/F)L, wherein one or more amino acids is substituted with a Lys-Ry; wherein the Ry comprises C18 octadecanedioic acid and a linker; and wherein one or more other amino acids are substituted with a Cys, Lys, Tyr, His, Ser, or Thr. [0026] In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. In some embodiments, the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a cysteine. In some embodiments, the peptide has the amino acid sequence VMAPRTLFL. In some embodiments, the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead. [0027] In some embodiments, the warhead is 8 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000011_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, - ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, - C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, - C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, - SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0028] In some embodiments, the warhead is
Figure imgf000011_0002
, wherein R2 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, 9 IPTS/128777599.4 Attorney Docket No.: CLS-039WO substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0029] In some embodiments, the warhead is
Figure imgf000012_0001
, wherein R3 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0030] In some embodiments, the warhead is
Figure imgf000012_0002
wherein R10 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently 10 IPTS/128777599.4 Attorney Docket No.: CLS-039WO selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0031] In some embodiments, the warhead is
Figure imgf000013_0001
, wherein X is a halogen. [0032] In some embodiments, the warhead is
Figure imgf000013_0002
. [0033] In some embodiments, the warhead is selected from the group consisting of
Figure imgf000013_0003
11 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000014_0001
[0034] In some embodiments, the warhead is conjugated to the Cys via the sulfur atom of the Cys. [0035] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000014_0002
, [0036] wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0037] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula 12 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000015_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0038] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000015_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0039] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000015_0003
, 13 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0040] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000016_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0041] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000016_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0042] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000016_0003
, 14 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker [0043] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000017_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0044] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000017_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0045] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula 15 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000018_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0046] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula O F
Figure imgf000018_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0047] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000018_0003
, 16 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0048] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000019_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0049] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000019_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0050] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula O
Figure imgf000019_0003
17 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. [0051] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000020_0001
F S O O , wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. In some embodiments, R is
Figure imgf000020_0002
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, - ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, - C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, - C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, - SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 18 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0052] In some embodiments, the synthetic peptide is an HLA-E-NKG2A complex specific inhibitor. [0053] In yet another aspect, the present disclosure provides a synthetic peptide comprising the amino acid sequence of VMAPRTLFL, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker; and further comprising one or more other amino acid substitution, In some embodiments, the one or more other amino acid substitution comprises: a. a substitution of the V residue at a position 1; b. a substitution of the M residue at a position 2; c. a substitution of the A residue at a position 3; d. a substitution of the P residue at a position 4; e. a substitution of the R residue at a position 5; f. a substitution of the T residue at a position 6; g. a substitution of the L residue at a position 7; h. a substitution of the F residue at a position 8; i. a substitution of the L residue at a position 9; j. or a combination of any of the foregoing substitutions. [0054] In some embodiments, the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAr. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. 19 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0055] In some embodiments, the linker comprises a PEG linker. In some embodiments, the linker comprises a PEG2 linker. In some embodiments, the linker comprises two PEG2 linkers. In some embodiments, the PEG2 linker is 8-amino-3,6-dioxaoctanoic acid. In some embodiments, the linker comprises a γ-Glu. [0056] In yet another aspect, the present disclosure provides a synthetic peptide comprising the formula
Figure imgf000022_0001
[0057] In yet another aspect, the present disclosure provides a synthetic peptide as disclosed herein comprising one or more additional modifications selected from: a. acetylated, formylated, propanoylated, hexanoylated, or myristoylated N-terminus; b. amidated C-terminus; c. substitution of one or more L-amino acid with a D-amino acid; d. substitution of one or more amino acid with a methyl-amino acid; and e. substitution of an α-amino acid with a β-amino acids. [0058] In yet another aspect, the present disclosure provides a peptide or peptidomimetic as disclosed herein. In some embodiments, the present disclosure provides a peptide or peptidomimetic comprising the amino acid sequence of any one of Tables 3-19. 20 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0059] In some embodiments, the present disclosure provides a peptide or peptidomimetic as disclosed herein, comprising one or more amino acid substitutions for reducing interaction of HLA-E with CD94/NKG2A, optionally relative to a comparator or the unmodified counterpart. In some embodiments, the present disclosure provides a peptide or peptidomimetic as disclosed herein that reduces interaction of HLA-E with CD94/NKG2A, optionally relative to a comparator or the unmodified counterpart. In some embodiments, the present disclosure provides a peptide or peptidomimetic as disclosed herein, comprising one or more amino acid substitutions for increasing NK-mediated cell killing, optionally as measured by a luciferase- based assay, optionally relative to a comparator or the unmodified counterpart. In some embodiments, the present disclosure provides a peptide or peptidomimetic as disclosed herein that increases NK-mediated cell killing, optionally as measured by a luciferase-based assay, optionally relative to a comparator or the unmodified counterpart. In some embodiments, the present disclosure provides a peptide or peptidomimetic as disclosed herein, lipidated to increase stability, optionally relative to a comparator or the unmodified counterpart. [0060] In some embodiments, a synthetic peptide or peptidomimetic as disclosed herein comprises an alanine substitute at position 1 or position 7. [0061] In some embodiments, a synthetic peptide or peptidomimetic as disclosed herein capable of displacing VL9 or VL9*5FAM from the antigen-binding groove of HLA-E. In some embodiments, the synthetic peptide or peptidomimetic has higher displacement efficiency at the antigen-binding groove of HLA-E relative to a comparator or the unmodified counterpart, optionally as analyzed by LC-MS or fluorescence polarization assays. [0062] In yet another aspect, the present disclosure provides a synthetic peptide/HLA-E complex, wherein the peptide is selected from a synthetic peptide as disclosed herein. In some embodiments, the synthetic peptide and the HLA-E in the complex are covalently linked. In some embodiments, the HLA-E is human HLA-E. In some embodiments, the synthetic peptide is covalently linked to amino acid residue Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E. In some embodiments, the synthetic peptide is covalently linked to an amino acid residue selected from the group of Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln- 72, Asn-77, Thr-80, Tyr-84, Trp-97, His-99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser- 147, Glu-152, His-155, Gln-156, Tyr-159, Thr-163, Cys-164, Trp-167, and Tyr-171 of human HLA-E. In some embodiments, the complex is inhibited in binding of CD94/NKG2A or prevents activation of CD94/NKG2A. 21 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0063] In yet another aspect, the present disclosure provides a pharmaceutical composition, comprising a synthetic peptide as disclosed herein and a pharmaceutically acceptable salt or carrier. BRIEF DESCRIPTION OF THE DRAWINGS [0064] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0065] FIG. 1A depicts HLA-E loaded with VL9 on cancer cells binding to CD94–NKG2A on NK cells and preventing a cytolytic attack by the NK cell. FIG. 1B shows a top view of VL9 binding the groove formed by two α-helices of HLA-E. Met-2 and Leu-9 anchor the 9-mer peptide in the binding groove. FIG. 1C depicts the design of a mixed 9- and 10-mer library based on VL9 including three different sets of amino acids. FIG. 1D depicts non-canonical amino acids used in the design of a combinatorial library. FIG. 1E depicts the complex of [HLA-E/VL9/B2M] immobilized on magnetic beads is subjected to affinity selections with the peptidomimetic library to identify putative binders through tandem mass spectrometry for synthesis and validation. [0066] FIGs. 2A-2F show the chemical formulas for peptides B1-B5 (SEQ ID NOs: 5-9) and B5 scrambled. FIG. 2G shows hit peptides B1–B5 containing positively charged residues (*) at the C- and N-termini and hydrophobic residues (#) in the core region. [0067] FIGs. 3A-3F show the chemical formulas for peptides B9 to B8 and a scrambled control peptide. [0068] FIG. 4 illustrates the results of a peptide exchange experiment of HLA-E/VL9 with exemplary peptides B1, B2, B3, B4, B5, B9, B10, B6, B7 and B8. [0069] FIG. 5A shows peptides B6–B10. Nle, Aoa, and Cha represent alternative amino acids of aliphatic Leu, whereas Nle and Mox were used to substitute the oxidation-sensitive thioether in Met. FIG. 5B shows some non-natural amino acids that can be single substitutions of Met2 or Leu9. FIG. 5C illustrates the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B1, B2, B3, B4, B5, and B9). FIG. 5D depicts the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B5, B9, B10, B6, B7 and B8). FIG. 5E depicts the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B5, B9, B5scrambled). FIG. 5F shows that all analogs of B5 displayed a high degree of exchange with HLA-E (low VL9 abundance in 22 IPTS/128777599.4 Attorney Docket No.: CLS-039WO intact HLA-E). FIG. 5G shows Peptides B6–B10 showed potent inhibition of CD94–NKG2A binding by BLI, whereas B9 scrambled displayed no inhibition. FIG. 5H shows Peptides B6– B10 induced loss of fluorescence polarization (measured in mP) over 4 h, whereas B9 scrambled and DMSO displayed no change of fluorescence polarization. FIG. 5I includes a plot depicting the thermal stability of the HLA-E/B2M complex loaded with the indicated peptides. FIG. 5J shows the melting temperature (Tm) shift for indicated alanine-scan peptide derivatives of B9 relative to B9. [0070] FIG. 6A illustrates a chemical reaction for installing an electrophilic aryl sulfonyl fluoride warhead by Pd-mediated coupling using an oxidative addition complex. FIG. 6B illustrates a reaction for cross-linking of electrophilic analogs of VL9 to HLA-E. FIG. 6C illustrates the results for exemplary cross-linking reactions with the electrophilic analogs of VL9 as measured my Mass Spectrometry (MS). FIG. 6D illustrates nucleophilic residues in a crystal structure on HLA-E situated within 6 Å from the binding groove of VL9 (highlighted in yellow). [0071] FIG. 7 illustrates an exemplary synthesis of Palladium Oxidative Addition Complex, (RuPhos)Pd(m-benzenefluorosulfonyl)Br, 1. [0072] FIG. 8A illustrates an exemplary peptide with a warhead (B9_8*) that is a covalent binder selective for HLA-E and inhibits binding of CD94/NKG2A. B9 was equipped with an electrophilic warhead at position 8 for covalent binding. FIG. 8B illustrates the results of a BLI experiment of binding of CD94/NKG2A to exemplary loaded HLA-E peptides (B9_8* and VL- 9_8*). FIG. 8C shows the results of crosslinking experiments measured MS of exemplary peptide/HLA-E complexes (grey traces represent protein reference spectra prior to incubation). [0073] FIG. 9 illustrates a synthesis schematic for making a candidate lipidated peptide. [0074] FIGs. 10A and 10B illustrate the structure, physical properties, and chromatogram results for the indicated lipidated peptide and its scrambled control, respectively. [0075] FIG. 11 illustrates the LC-MS plots from B9 and lipidated B9 were incubated in 25% human serum or 10% FBS for 24 hours, showing that the lipidation increases B9’s stability in serum-containing media. [0076] FIG. 12 illustrates the indicated lipidated B9 peptide and control exchange rates with VL9. The exchange rate of B9 is not impaired by lipidation in PBS, and is enhanced in FBS- containing media relative to B9. [0077] FIG. 13 illustrates the indicated lipidated B9 peptide and control B9 BLI inhibition are similar in PBS, but lipidated B9 outperforms B9 in FBS-containing media. 23 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0078] FIGs. 14A-14G show the interaction of HLA-E with CD94–NKG2A can be inhibited by peptides discovered from a focused library. FIGs. 14A-14B show how HLA-E/B2M/VL9 (1 μM) was incubated with 10-fold excess of hit peptidomimetics (10 μM) for ligand exchange and subsequently loaded onto biosensors to measure association and dissociation of CD94–NKG2A (200 nM). FIG. 14C shows the binding of CD94–NKG2A was inhibited by peptides B2, B3, B5, after incubation with HLA-E as determined by BLI. FIG. 14D shows how the abundance of VL9 on intact HLA-E complexes was determined by LC-MS after ligand exchange. FIG. 14E shows VL9 abundance was the lowest after B5 exchange for 16 h at room temperature. FIG. 14F shows how the displacement of fluorescently labeled VL9*5FAM from the complex with HLA- E and B2M by the hit sequences was monitored by changes in fluorescence polarization (ΔmP). FIG. 14G shows Peptides B1, B2, B3, and B5 led to changes in fluorescence polarization (measured in mP) over 4 h. [0079] FIGs. 15A-15F show the crystal structure of HLA-E/B2M/B9. FIG. 15A: Overall structure. HLA-E and B2M are shown as surface and cartoon representations and B9 is shown as sticks. FIG. 15B: Detailed interactions between HLA-E and B9. Arg-62 from the NKG2A/CD94/HLA-E/B2M/VL9 co-receptor structure is shown as transparent green sticks. Hydrogen bonds are shown as black, dashed lines. FIG. 15C: Alignment of the B9-bound HLA- E/B2M structure (with only B9 peptide shown) with the NKG2A/CD94/HLA-E/B2M/VL9 co- receptor structure (with only protein shown) reveals several clashes. FIG. 15D: Alignment of the HLA-E/B2M/B9 structure with the HLA-E/B2M/Mtb44 peptide-bound structure. [0080] FIGs. 16A-16H show that B9 outperforms Mtb44 (a known inhibitory peptide) in biophysical assays. FIG. 16A: Exchange of B9 with HLA-E/VL9*5FAM was compared to Mtb44 with a higher degree of VL9 exchange via LC-MS. FIG. 16B: Validation of exchange was shown through loss of polarization with VL9, B9, and Mtb44 showing similar exchange kinetics in comparison to their scrambled counterparts. FIG. 16C: BLI demonstrated a stronger loss of binding with CD94-NKG2A than Mtb44. FIG. 16D: B9, VL9, Mtb44 or scrambled analogs thereof bearing a fluorescent label were incubated with HLA-R/B2M/VL9 to monitor changes of FP over 2 h indicating binding of the fluorescent peptide to HLA-E. FIG. 16H: The strongest gain of FP was observed for B9*5FAM, whereas the scrambled sequence analogs showed no change of FP over 2 h. [0081] FIGs. 17A-17F shows inhibitory peptide B9 led to HLA-E stabilization and loss of CD94-NKG2A binding on cells. FIG. 17A: HLA-E (3D12-APC) staining of SCaBER cells after VL9 pulse and peptide exchange. Plotted medians on the graph to the right of the histograms. FIG. 17B: HLA-E/VL9 (tetramerized CD94-NKG2A/StrepAF647) staining of SCaBER cells 24 IPTS/128777599.4 Attorney Docket No.: CLS-039WO after VL9 pulse and peptide exchange. Plotted medians on the graph to the right of the histograms. FIG. 17C: HLA-E (3D12-APC) staining of the competition of peptides with VL9 at ratiometric levels (total peptide concentration of 20uM). Corresponding medians plotted on the graph to the right. FIG. 17D: HLA-E/VL9 (tetramerized CD94-NKG2A/StrepAF647) staining of the competition of peptides with VL9 at ratiometric levels (total peptide concentration of 20uM). Corresponding medians plotted on the graph to the right. FIG. 17E: Jurkat reporter stimulation after pulse and exchange with peptides at 10uM and 20uM exchanged peptides. Luminescence was measured as a report on Jurkat interaction with cells. Comparison to surface levels of HLA-E stained with 3D12-APC and analyzed via FACS. FIG. 17F shows Lucia® luciferase secretion, as measured in NFAT-Lucia reporter system, for VL9, B9, and Mtb44 groups. DETAILED DESCRIPTION [0082] The present disclosure is based, in part, upon the development of synthetic peptides and peptidomimetics that bind HLA-E in a covalent or non-covalent manner to form peptide-HLA-E complexes. Additionally, the peptide-HLA-E complexes can modulate or inhibit the binding of HLA-E to its cognate receptor CD94/NKG2A or prevent activation of CD94/NKG2A. The synthetic peptides and peptidomimetics can be used to modulate or abrogate HLA- E/CD94/NKG2A signaling in NK and T cells. [0083] Various components and aspects of the disclosure are described in further detail in the subsections below. I. Definitions [0084] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Mention of techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. [0085] Throughout the description, where compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present invention that consist essentially of, or consist of, the recited 25 IPTS/128777599.4 Attorney Docket No.: CLS-039WO components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. [0086] In the disclosure, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. [0087] Further, it should be understood that elements and/or features of a composition or a method provided and described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure and invention(s) herein, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and/or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of invention(s) provided, described, and depicted herein. [0088] As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value. [0089] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element. [0090] The term “and/or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise. [0091] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” 26 IPTS/128777599.4 Attorney Docket No.: CLS-039WO in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context. [0092] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “is,” “are” or any other variation thereof, are intended to cover a non-exclusive inclusion. They are to be interpreted synonymously with the phrases “having at least” or “including at least”. The term “consisting of” refers to including, and being limited to, whatever follows the phrase “consisting of.” [0093] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context. [0094] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously. [0095] At various places in the present specification, variable or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. [0096] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention(s) unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure. [0097] As used herein, “residue” refers to a position in a protein and its associated amino acid identity. [0098] As used herein, “Natural Killer cell” or “NK cell” refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56, and/or CD57, the absence of the alpha/beta or gamma/delta T- 27 IPTS/128777599.4 Attorney Docket No.: CLS-039WO cell receptor (TCR) complex on the cell surface, the ability to bind to and kill cells that fail to express “self MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK-activating receptors, and the ability to release cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art such as fluorescence assisted cell sorting (FACS). [0099] As used herein, “NKG2A” or “KLRC1” refers to the full length NKG2A. NKG2A (OMIM 161555, the entire disclosure of which is herein incorporated by reference) is a member of the NKG2 group of transcripts (see e.g., Houchins, et al. (1991), The Journal of experimental medicine). NKG2A and NKG2C form heterodimer receptors with CD94 and both target the same p/HLA-E complex, but ligation with the p/HLA-E complex induces an inhibitory signal for NKG2A and an activation signal for NKG2C. In contrast to the classical HLA class I molecules that present antigenic peptide epitopes to the TCR in complex with CD3, HLA-E presents a limited set of conserved signal peptides to NKG2A and NKG2C. These peptides bound and presented by HLA-E are derived from leader sequences of classical HLA class I molecules. The receptor dimer CD94/NKG2A found on natural killer (NK) cells recognizes these short peptides bound to human leukocyte antigen-E (HLA-E), which has an inhibitory effect on NK cells. The peptide–HLA-E complex is expressed in most human tissues as a marker of cell health and protects cells from the cytotoxic activation of NK cells. The expression of HLA-E has also been associated with different types of cancer as a mechanism to evade attacks by NK cells. [0100] The terms “major histocompatibility complex” and “MHC” also refer to the polymorphic glycoproteins encoded by the MHC class I or class II genes, where appropriate in the context, and proteins comprising variants thereof that bind T cell epitopes (e.g., class I or class II epitopes). Such proteins are also referred to as “MHC molecule” or “MHC protein” herein. The terms “MHC class I” or “MHC I” are used interchangeably to refer to protein molecules comprising an α chain composed of three domains (α1, α2 and α3), and a second, invariant β2-microglobulin. The α3 domain is linked to the transmembrane domain, anchoring the MHC class I molecule to the cell membrane. Antigen-derived peptide epitopes, which are located in the peptide-binding groove, in the central region of the α1/α2 heterodimer. MHC Class I molecules such as HLA-A, HLA-B, HLA-C, and HLA-E are part of a process that presents short polypeptides to the immune system. These polypeptides are typically 8-11 amino acids in length and originate from proteins being expressed by the cell, which can be endogenous proteins or exogenous proteins (e.g., viral or bacterial proteins, vaccine proteins). MHC class I 28 IPTS/128777599.4 Attorney Docket No.: CLS-039WO molecules present antigen to CD8+ cytotoxic T cells. Histocompatibility leucocyte antigen E (HLA-E), is a conserved nonclassical HLA class I molecule that binds a limited peptide repertoire. Antigens delivered endogenously to APCs are processed primarily for association with MHC class I. Antigens delivered exogenously to APCs are processed primarily for association with MHC class II. As used herein, MHC proteins (MHC Class I or Class II proteins) also includes MHC variants which contain amino acid substitutions, deletions or insertions and yet which still bind MHC peptide epitopes (MHC Class I or MHC Class II peptide epitopes). The term “MHC,” “MHC molecule,” or “MHC protein” also includes an extracellular fragment of a full-length MHC protein that retains the ability to bind the cognate epitope, for example, a soluble MHC. As used herein, the term “soluble MHC” refers to an extracellular fragment of a MHC comprising corresponding α1 and α2 domains that bind a class I T cell epitope or corresponding α1 and β1 domains that bind a class II T cell epitope, where the α1 and α2 domains or the α1 and β1 domains are derived from a naturally occurring MHC or a variant thereof. The classical MHC class I (termed “Ia”) molecules (HLA-A, HLA-B and HLA-C) are highly polymorphic and are ubiquitously expressed on most somatic cells. In contrast, non classical MHC class I (termed “Ib”) molecules (HLA-E, HLA-F and HLA-G) are broadly defined by a limited polymorphism and a restricted pattern of cellular expression. [0101] The term “HLA-E” refers wild type, full length HLA-E. Among class Ib molecules, HLA-E is characterized by a low polymorphism and a broad mRNA expression on different cell types. Lee et al. (1988) J Immunol. 160:4951- 60. HLA-E is nonpolymorphic with only two functional alleles present in the human population: the HLA-E*01:01 and the HLA-E*01:03 variants. These two alleles only differ in a single amino acid at position 107, being arginine (01:01) or glycine (01:03). This class I molecule is a heterodimer consisting of a heavy chain and a light chain (β2-microglobulin, β2m, B2M). The heavy chain is approximately 45 kDa and its gene contains 8 exons. Cell surface expression of HLA-E requires the availability of β2- microglobulin (Ulbrecht et al. (1999) Eur J Immunol. 29:537-47) and of a set of highly conserved nonameric peptides derived from the leader sequence of various HLA class I molecules including HLA-A, -B, -C, and -G (see e.g., Braud et al. (1997) Eur J Immunol. 27: 1164-9; Ulbrecht et al. (1998) J Immunol. 160:4375-85). HLA-E binds NK cells and some T cells, binding specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C, and not to the inhibitory KIR receptors. See, e.g., Braud et al. (1998) Nature 391 :795-799. Surface expression of HLA-E is sufficient to protect target cells from lysis by CD94/NKG2A+ NK cell clones. 29 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0102] The term “MHC protein” also includes MHC proteins of non-human species of vertebrates. MHC proteins of non-human species of vertebrates play a role in the examination and healing of diseases of these species of vertebrates, for example, in veterinary medicine and in animal tests in which human diseases are examined on an animal model, for example, experimental autoimmune encephalomyelitis (EAE) in mice (mus musculus), which is an animal model of the human disease multiple sclerosis. Non-human species of vertebrates are, for example, and more specifically mice (mus musculus), rats (rattus norvegicus), cows (bos taurus), horses (equus equus) and green monkeys (macaca mulatta). MHC proteins of mice are, for example, referred to as H-2-proteins, wherein the MHC class I proteins are encoded by the gene loci H2K, H2L, and H2D and the MHC class II proteins are encoded by the gene loci H2I. [0103] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” [0104] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom. Such progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. [0105] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. [0106] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder. [0107] The term “modulation” refers to an increase or decrease in the level of a target molecule or the function of a target molecule. The term “modulator” as used herein refers to 30 IPTS/128777599.4 Attorney Docket No.: CLS-039WO modulation of (e.g., an increase or decrease in) the level of a target molecule or the function of a target molecule. Chemical Definitions [0108] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987. [0109] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. [0110] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw–Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. 31 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0111] As used herein, the term “kd” (s-1) refers to the dissociation rate constant between a given entity and a target (e.g., of a particular peptide-target interaction). This value is also referred to as the koff value. [0112] As used herein, the term “ka” (M-1×s-1) refers to the association rate constant of a given entity and a target (e.g., a particular peptide-target interaction). This value is also referred to as the kon value. [0113] As used herein, the term “KD” (M) refers to the dissociation equilibrium constant of a given entity and a target (particular interaction between an entity and its target (e.g., a particular peptide-target interaction)). KD = kd/ka. [0114] As used herein, the term “KA” (M-1) refers to the association equilibrium constant of a given entity and a target (e.g., a particular polypeptide-target interaction). KA = ka/kd. [0115] The affinity of a molecule X for its target Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are as described above. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). [0116] As used herein, the term “lipidated” refers to a synthetic peptide or peptidomimetic comprising a lipid moiety (e.g., C18 octadecanedioic acid group). Without being limited to the mechanism of action, lipidation of peptides can be used to increase, for example, the pharmacokinetic properties, improve metabolic stability, reduce enzymatic degradation, lower excretion and metabolism, in vivo half-life, etc. [0117] As used herein, in some embodiments, the terms “B5.1” and “B9” are used interchangeably. As used herein, in some embodiments, the terms “B5.2” and “B10” are used interchangeably. As used herein, in some embodiments, the terms “B5.3” and “B6” are used interchangeably. As used herein, in some embodiments, the terms “B5.4” and “B7” are used interchangeably. As used herein, in some embodiments, the terms “B5.5” and “B8” are used interchangeably. [0118] As used herein a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, 32 IPTS/128777599.4 Attorney Docket No.: CLS-039WO in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound. [0119] In the compositions provided herein, an enantiomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure R–compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R–compound. In certain embodiments, the enantiomerically pure R– compound in such compositions can, for example, comprise, at least about 95% by weight R– compound and at most about 5% by weight S–compound, by total weight of the compound. For example, a pharmaceutical composition comprising enantiomerically pure S–compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S–compound. In certain embodiments, the enantiomerically pure S– compound in such compositions can, for example, comprise, at least about 95% by weight S– compound and at most about 5% by weight R–compound, by total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier. [0120] When a range of values is listed, it is intended to encompass each value and sub–range within the range. For example, “C1-C6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4- C5, and C5-C6 alkyl. [0121] “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1– naphthyl and 2–naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each 33 IPTS/128777599.4 Attorney Docket No.: CLS-039WO instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is substituted C6-C14 aryl. [0122] “Halo” or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom. The term “halide” by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom. In certain embodiments, the halo group is either fluorine or chlorine. [0123] As used herein, the term “linker” refers to either a bond or a moiety which at one end exhibits a grouping able to enter into a covalent bonding with the residue or a reactive functional group of the synthetic peptide or peptidomimetic, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding, and bridges, either directly or indirectly the synthetic peptide or peptidomimetic and the lipid moeity, e.g. C18 octadecanedioic acid group. Those of ordinary skill in the art may use the term spacer and linker interchangeably. In some embodiments, the linker is at least one polyethylene glycol polymer. [0124] In some embodiments, the linker attached to a Lysine residue in the peptide or peptidomimetic. In some embodiments, the linker can be at a primary amine (-NH-)2 present at the N-terminus of each polypeptide chain (referred to as α -amine) or the side chain of the lysine (Lys, K) residue (referred to as E-amine). In some embodiments, a linker can be attached to a peptide compound at a sulfhydryl group (-SH): this group is present in the side chain of cysteine (Cys, C). Typically, cysteines are linked together between their side chains by disulfide bonds (- S-S-) as part of the secondary or tertiary structure of the protein. These must be reduced to a thiol group to make them available for most types of reactive groups to crosslink. In some embodiments, the linker can be attached to the peptide compound at a carbonyl (-CHO): by oxidation of post-translational modifications (glycosylation) of polysaccharides with sodium metaperiodate, ketone or aldehyde groups can be generated in glycoproteins. For example, the linker can be a cleavable linker. For example, the linker can be a non-cleavable linker. [0125] As used herein, the term “polyethylene glycol” or PEG refers to a polymer containing ethylene glycol monomer units of formula —O—CH2—CH2—. In some embodiments, suitable polyethylene glycols may have a free hydroxy group at each end of the polymer molecule or may have one hydroxy group etherified with a lower alkyl, e.g., a methyl group. Also suitable are derivatives of polyethylene glycols having esterifiable carboxy groups. Polyethylene glycols 34 IPTS/128777599.4 Attorney Docket No.: CLS-039WO useful in the present disclosure can be polymers of any chain length or molecular weight and can include branching. [0126] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention(s) unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure. [0127] This present disclosure relates to International Application No. PCT/US2023/022979, filed on May 19, 2023, and U.S. Patent Application No. 18/952,930, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. II. Peptides and Peptidomimetics [0128] Disclosed herein are lipidated synthetic peptides, peptidomimetics, and libraries thereof. In some embodiments, the synthetic peptides and peptidomimetics are a peptide antigen bound to and presented by the MHC class I molecule major histocompatibility complex E (HLA-E). [0129] Human leukocyte antigen E (HLA-E) belongs to the family of non-classical major histocompatibility complex (MHC) class Ib molecules. In the population, HLA-E displays limited polymorphism with two dominant alleles, HLA-E*01:01 and HLA-E*01:03, differing by only one amino acid which are detected in over 99% of the population.1-3 HLA-E, in complex with beta-2 microglobulin (B2M), is expressed on the cell surface of nucleated cells with broad tissue distribution and typically low expression levels(Wei, X., and Orr, H. T. (1990) Human Immunology). Its primary function is the presentation of antigens for immune surveillance (Braud, V. M. et al. (1998) Nature; Lee, N., Llano, M., Carretero, M., Ishitani, A., Navarro, F., López-Botet, M., and Geraghty, D. E. (1998) HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A, Proceedings of the National Academy of Sciences ). In homeostasis, HLA-E binds signal peptides derived from the leader sequences of MHC class I molecules HLA-A, B, C, or G as a sign of cellular health. These signal peptides are collectively referred to as “VL9,” and are nonameric peptides of the general sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1). (Braud, V., et al. (1997) European Journal of Immunology; O'Callaghan, C. A., et al. (1998) Molecular Cell; Llano, M et al. (1998) European Journal of Immunology). The HLA-E/B2M/VL9 complex is recognized by the inhibitory 35 IPTS/128777599.4 Attorney Docket No.: CLS-039WO receptor heterodimer CD94–NKG2A of natural killer (NK) cells to prevent a cytolytic response (FIG. 1A) (Braud (1998) Nature). [0130] In addition, HLA-E receptors have been shown to present other foreign and stress-related antigens to αβ receptors on CD8+ T-cells. For certain types of pathogens (e.g., cytomegalovirus), HLA-E-restricted CD8+ T-cells represent a considerable component of the adaptive immune response, highlighting the potential of this protein target for vaccine development (Walters, L. C., et al. (2018) Nature Communications; Voogd, L., et al. (2022) Trends in Immunology; Hansen, S. G.et al. (2016) Science; Hansen, S. G., et al. (2013) Science). [0131] Analysis of the interface between HLA-E and CD94–NKG2A from previously published structures highlights the native VL9 peptide ligand as a key modulatory unit for this protein– protein interaction (PPI). For the present disclosure, the inventors leveraged available structural and functional information for the discovery of non-natural peptide ligands that retain binding to HLA-E but abrogate interactions with CD94–NKG2A, in part, through the use of affinity selection–mass spectrometry (AS-MS). [0132] In some aspects of the disclosure, the peptide or peptidomimetic has the amino acid sequence of an antigen. Peptide antigens comprise, but are not limited to peptides that have the amino acid sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1), ( “VL9”), derived from signal peptides of the MHC class I molecules HLA-A, -B, -C, and -G. In some embodiments, the peptide or peptidomimetic is based on the ligand for the NKG2A/CD94 inhibitory receptor in mice, the nonclassical MHC molecule Qa-1b, the mouse HLA-E ortholog, which presents the peptide AMAPRTLLL (SEQ ID NO: 355), referred to as “Qdm” (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. In some embodiment, these are comparators. In some embodiments, a comparator is fluorescent analog of VL9 bearing a fluorescein at position 5 (i.e., VL9*5FAM). [0133] In some embodiments a comparator is Mtb44. Mtb44 (having the amino acid sequence of RLPAKAPLL) is a peptide derived from the pathogenic Mycobacterium tuberculosis. Mtb44 is a well-characterized binder of HLA-E with comparable binding affinity as endogenous VL9 and has demonstrated ability to inhibit coreceptor interaction. [0134] In some embodiments the peptide sequence comprises the amino acid sequence VMAPRTLVL (SEQ ID NO: 2). In some embodiments, the peptide is 8, 9, or 10 amino acids long. In some embodiments one or more amino acids of the VL9 sequence are substituted. In some embodiments, the substitution is a substitution of the V residue at position 1 (Val1), the M 36 IPTS/128777599.4 Attorney Docket No.: CLS-039WO residue at position 2 (Met2), the A residue at position 3 (Ala3), P residue at position 4 (Pro4), R residue at position 5 (Arg5), the T residue at position 6 (Thr6), the L residue at position 7 (Leu7), the F residue at position 8 (Phe8), the L residue at position 9 (Leu9), or a combination of any of the foregoing substitutions. In some embodiments, the anchor residues Met2 and Leu9 are constant. In some embodiments, the residue at position 10 is a Lys. In some embodiments, the R residue at position 5 is substituted with aliphatic and aromatic monomers, and the F residue at position 8 is substituted by polar and charged residues. [0135] In some embodiments, the peptide antigen comprises, but are not limited to peptides that have the amino acid sequence VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO: 1), (referred to as VL9), derived from signal peptides of the MHC class I molecules HLA-A, -B, -C, and -G. In some embodiments, the substituted amino acid is a canonical amino acid. Canonical amino acids for use in substitutions are listed in TABLE 1. In some embodiments, the canonical substituted amino acids are an Ala, a Ser, a Gln, or an Arg. TABLE 1 Canonical amino acids used in the peptides and peptidomimetics. TABLE 1
Figure imgf000039_0001
[0136] In some aspects of the disclosure, the synthetic peptide or peptidomimetic comprises one or more of 43 non-canonical amino acids. In some embodiments, amino acids in VL9 are 37 IPTS/128777599.4 Attorney Docket No.: CLS-039WO substituted with non-canonical amino acids. Non-canonical amino acids that can be used for substitution are shown in TABLE 2. [0137] TABLE 2 Non-canonical amino acids for use in the peptides and peptidomimetics. TABLE 2
Figure imgf000040_0001
38 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000041_0001
[0138] In some embodiments, substitutions in the VL9 peptide comprise the non-canonical amino acids 4Af, hAr, Dff, Msn, or Cha. In some embodiments the amino acids of the synthetic peptide are mixed canonical and non-canonical amino acids. [0139] In some instances, hAr and hArg are used interchangeably. [0140] In some embodiments, the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAR. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. [0141] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises X1-Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3) or X1-Met-X3-X4-X5-Ala-X7-X8- Leu-Lys (SEQ ID NO: 4), wherein each of the X1, X3, X4, and X7 are independently selected from Gly, Ala, Met, Pro, Cpa, Cha, Ser, Asn, Gln, Msn, Phe, Tyr, His, Trp, 4Py, 4Af, Tha, Dff, Asp, Glu, Lys, Arg, hAr, or Aad; X5 is Gly, Ala, Val, Leu, Met, Pro, Cpa, Cba, Cha, Aoa, Phe, Trp, Mff, Dff, Tff, Tha, Nal, hPh, Dmf, Php, or Amb; and X8 is Ser, Thr, Asn, Gln, Msn, Hyp, Asp, Glu, Lys, Arg, Dab, Orn, Aad, or hAr. [0142] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises X1-Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3) or X1-Met-X3-X4-X5-Ala-X7-X8- 39 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Leu-Lys (SEQ ID NO: 4), wherein each of X1, X3, X4, and X7 are independently selected from an amino acid of mixed polarity. [0143] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises X1-Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3) or X1-Met-X3-X4-X5-Ala-X7-X8- Leu-Lys (SEQ ID NO: 4), wherein X5 is an apolar amino acid. [0144] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises X1-Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3) or X1-Met-X3-X4-X5-Ala-X7-X8- Leu-Lys (SEQ ID NO: 4), wherein X8 is a polar amino acid. [0145] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises a modified side chain. Exemplary side chains that can be used to modify the peptide or peptidomimetic of the disclosure are listed in TABLE A. TABLE A
Figure imgf000042_0001
40 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000043_0001
[0146] In some embodiments, amino acid sequence of the peptide or peptidomimetic is selected form the amino acid sequences in TABLE 3. In some embodiments, the substituted peptide is selected from the group of SEQ ID NOs: 5-9. [0147] TABLE 3 exemplary amino acid sequences of peptidomimetics disclosed herein. TABLE 3
Figure imgf000043_0002
[0148] In some embodiments, additional amino acids in SEQ ID NOs: 3-9 are substituted. In some embodiments one or more amino acid is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substituted amino acid is at position 3 or position 8. In some embodiments, the substituted amino acid is at position 8 and is substituted with a Cys. In some embodiments, the substituted amino acid is at position 3 and is substituted with a Cys. Exemplary Cys substituted peptides and peptidomimetic sequences are listed in TABLE 4. In some embodiments, the Cys substituted peptide is selected from the group of SEQ ID NOs: 10- 19. [0149] In some embodiments, terminal NH2- refers to the N-terminus within the adjacent listed amino acid. For example, the “NH2-” in “NH2-Ala-” refers to N-terminus within the alanine residue. In some embodiments, a terminal “-OH” or “-COOH” refers to the C-terminus within the adjacent listed amino acid. For example, the “-OH” in “Leu-OH” refers to the C-terminus within the leucine residue. 41 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0150] TABLE 4 exemplary amino acid sequences of peptidomimetics disclosed herein. TABLE 4
Figure imgf000044_0001
[0151] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises hAr-X2-hAr-Gln-Mff-A-Cha-hAr-X9 (SEQ ID NO: 20) wherein X2 is Nle or Mox; and X9 is Leu, Aoa, or Cha. [0152] TABLE 5 exemplary amino acid sequences of peptidomimetics disclosed herein. TABLE 5
Figure imgf000044_0002
[0153] In some embodiments, additional amino acids in SEQ ID NOs: 21-25 are substituted. In some embodiments one or more amino acid is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substituted amino acid is at position 3 or position 8. In some embodiments, the substituted amino acid is at position 8 and is substituted with a Cys. In some embodiments, the substituted amino acid is at position 3 and is substituted with a Cys. 42 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Exemplary Cys substituted peptides and peptidomimetic sequences are listed in TABLE 4. In some embodiments, the Cys substituted peptide is selected from the group of SEQ ID NOs: 26- 34. [0154] TABLE 6 lists exemplary amino acid sequences of peptidomimetics disclosed herein. TABLE 6
Figure imgf000045_0001
[0155] In some aspects of the disclosure, the amino acid sequence of the peptide or peptidomimetic comprises VMAPRTLFL (SEQ ID NO:36) or VMAPRT(L/V)(V/L/I/F)L with one or more amino acid substitutions. In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises wherein one or more amino acids that are substituted with a Cys, Lys, Tyr, His, Ser, or Thr. [0156] In some embodiments, the amino acid sequence of the peptide or peptidomimetic comprises a substitution of the V residue at a position 1; a substitution of the M residue at a position 2; a substitution of the A residue at a position 3; a substitution of the P residue at a position 4; a substitution of the R residue at a position 5; a substitution of the T residue at a position 6; a substitution of the L residue at a position 7; a substitution of the F residue at a position 8; a substitution of the L residue at a position 9; or a combination of any of the foregoing substitutions. In some embodiments, the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 4 and the amino 43 IPTS/128777599.4 Attorney Docket No.: CLS-039WO acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. In some embodiments, the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAR. In some embodiments, the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. [0157] In another aspect of the disclosure, the synthetic peptides and peptidomimetics are designed to bind HLA-E. In some embodiments, the synthetic peptides and peptidomimetics bind HLA-E with low, medium, or high affinity. In some embodiments, the synthetic peptides and peptidomimetics bind HLA-E with higher affinity than VL9. In some embodiments, the synthetic peptides and peptidomimetics are covalently bound to HLA-E. In some embodiments, the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln-72, Asn-77, Thr-80, Tyr-84, Trp-97, His-99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser-147, Glu-152, His-155, Gln- 156, Tyr-159, Thr-163, Cys-164, Trp-167, or Tyr-171 of human HLA-E. In some embodiments, the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E. [0158] In another aspect of the disclosure, the synthetic peptides and peptidomimetics are listed in TABLE 13, TABLE 14, TABLE 15, TABLE 16, TABLE 17, TABLE 18, or TABLE 19. Peptide Libraries [0159] The inventors of the present disclosure demonstrated the ability to use AS-MS to discover novel peptide binders from libraries with up to 200 million synthetic members in a single experiment27-29 and enable the identification of peptidomimetic modulators of the interaction between HLA-E and CD94–NKG2A. In some embodiments, synthetic peptide libraries can be readily prepared by solid-phase peptide synthesis (SPPS) and allow the facile introduction of non-natural amino acids into a library design to extend the chemical diversity.30 In some embodiments, the resulting non-natural peptides can displace VL9 from the antigen- binding groove of HLA-E and inhibit its molecular recognition by CD94–NKG2A by design. In some embodiments, this can be validated by fluorescence polarization (FP), liquid chromatography-mass spectrometry (LC-MS), and biolayer interferometry (BLI) assays. In some embodiments, the peptides or peptidomimetics stabilize the HLA-E/B2M complex, as 44 IPTS/128777599.4 Attorney Docket No.: CLS-039WO determined, e.g., by a thermal shift assay. In some embodiments, a synthetic peptide or peptidomimetic as disclosed herein (e.g., peptide B9) can stabilize HLA-E on the surface of cells to a similar extent as a comparator peptide (e.g., exogenous VL9, Mtb44, etc.), optionally while leading to a dose-dependent loss of co-receptor engagement in a purified format and/or on reporter cells overexpressing CD94–NKG2A. [0160] Collectively, these results highlight the utility of non-natural amino acids, rational library design, and AS-MS in the discovery and development of peptidomimetics as a novel class of PPI modulating drugs. [0161] In some aspects, the disclosure is directed to libraries of synthetic peptides and peptidomimetics disclosed herein. In some embodiments, the peptide library has the design X1- Met-X3-X4-X5-Ala-X7-X8-Leu (SEQ ID NO: 3) or X1-Met-X3-X4-X5-Ala-X7-X8-Leu-Lys (SEQ ID NO: 4) is generated, wherein each of X1, X3, X4, and X7 are independently selected from Gly, Ala, Met, Pro, Cpa, Cha, Ser, Asn, Gln, Msn, Phe, Tyr, His, Trp, 4Py, 4Af, Tha, Dff, Asp, Glu, Lys, Arg, hAr, Aad; X5 is Gly, Ala, Val, Leu, Met, Pro, Cpa, Cba, Cha, Aoa, Phe, Trp, Mff, Dff, Tff, Tha, Nal, hPh, Dmf, Php, Amb; and X8 is Ser, Thr, Asn, Gln, Msn, Hyp, Asp, Glu, Lys, Arg, Dab, Orn, Aad, hAr. In some embodiments, the half of the library has a Lys at the C- terminus. In some embodiments, anchor residues Met2 and Leu9 are set constant in the library design. In some embodiments, Arg5 is substituted with aliphatic and aromatic monomers, and Phe8 is replaced by polar and charged residues. In some embodiments, 21 non-canonical amino acids (shown in TABLE 2) are included in the library design. [0162] In some embodiments the library is 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million peptides in size. In some embodiments the library is 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, or 100 million, or 200 million peptides in size. [0163] Determination of the crystal structure of, e.g., peptide B9 in complex with HLA-E/B2M confirmed the location of peptides (e.g., B9) in the antigen-binding groove of HLA-E with the expected anchor residues. Without wishing to be bound by theory or mechanism, the otherwise linear peptide B9, as disclosed herein, can adopt a characteristic kinked conformation of its backbone presenting functional groups to the solvent-exposed surface intended to inhibit interactions with CD94–NKG2A. Unexpectedly, it was found that binding of B9 to HLA-E resulted in partial denaturation of helix in the binding groove known to house key interacting residues with CD94-NKG2A. Lipidation 45 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0164] In some embodiments, each of the peptides disclosed herein are contemplated as lipidated peptides. In some embodiments, that lipidation involves the incorporation of a lipid moiety and spacer (e.g. linker) into the disclosed synthetic peptides or peptidomimetics. In some embodiments, that incorporation is such that one or more amino acids is substituted with a Lys- Ry; and wherein the Ry comprises C18 octadecanedioic acid and, optionally, a linker. [0165] In some embodiments, that incorporation is such that one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises a lipid. In some embodiments, the Ry comprises a hydrocarbon-based compound comprising two -COOH carboxyl functions. In some embodiments, the Ry comprises a saturated diacid. In some embodiments, the Ry comprises an unsaturated diacid.In some embodiments, Ry comprises ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, tetradecanedioic acid (C14 diacid), hexadecanedioic acid (C16 diacid), heptadecanedioic acid (C17 diacid), octadecanedioic acid (C18 diacid), nonadecanedioic acid (C19 diacid), eicosanodioic acid (C20 diacid), heneicosanodioic acid (C21 diacid) myristic acid, docosanodioic acid (C22 diacid), tetracosanedioic acid (C24 diacid)oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid (or 1,10-decanedioic acid), octadecamethylenedicarboxylic acid, eicosadicarboxylic acid, including branched or substituted derivatives thereof, or mixtures thereof. Non-limiting examples of unsaturated diacids include (Z)-butenedioic acid (maleic acid), (E)-butenedioic acid (fumaric acid), (Z and E)-pent-2-enedioic acid (glutaconic acid), 2- decenedioic acid, dodec-2-enedioic acid (traumatic acid), (2E,4E)-hexa-2,4-dienedioic acid (muconic acid), and mixtures or combinations thereof [0166] In some embodiments, Ry comprises a C14 diacid group, C16 diacid groups, or C18 diacid group. [0167] In some embodiments, Ry comprises a lipid moiety means for improving pharmacological properties, e.g. stability and half-life. [0168] The incorporation of lipid moieties into peptides can yield improved pharmacological attributes, encompassing heightened stability and subsequent extension of the drug’s in vivo half-life. The present disclosure demonstrates an efficacious method for altering the pharmacokinetic and pharmacodynamic characteristics of primary peptide therapeutics. The inclusion of lipid groups onto a peptide scaffold has the potential to significantly elevate enzymatic stability, receptor specificity, potency, bioavailability, and drug delivery capabilities. The inventors of the present disclosure investigated the influence of fatty acid group attributes, including length, composition, polarity, and steric demand, as well as the spacer type between 46 IPTS/128777599.4 Attorney Docket No.: CLS-039WO the active molecule and the lipid tail on the disclosed peptides. It was found that lipid moieties, such as C18 octadecanedioic acid group attached to Lys-26, in some embodiments, along with an extended spacer, e.g., composed of γ-Glu linked to two 8-amino-3,6-dioxaoctanoic acid groups, improved the pharmacological properties (e.g., stability and half-life) of the peptides. It was also identified that the lipidation of a residue in any one of the disclosed peptides, wherein the residue is solvent-exposed and not involved in any major binding interactions, can effectively improve the peptides or peptidomimetics pharmacological properties (e.g., stability and half-life). [0169] In some embodiments, a lipidated peptide or peptidomimetic has enhanced stability and bioactivity, e.g., in serum-containing media, relative to its unlipidated counterpart or a comparator. In some embodiments, lipid-modified B9 stabilizes HLA-E at the surface of target cells and leads to dose-dependent cytotoxic cell-killing by human NK cells. III. Modifications [0170] The central limitation in the development of peptide therapeutics is their short circulation time resulting from rapid enzymatic degradation and renal clearance. Methods to evade renal elimination by increasing the molecular weight have emerged, but extensive modifications can cause undesired steric hindrance during target binding. For small molecules, an alternative approach to modulate pharmacokinetic profiles and improve the potency and selectivity of a potential drug is the exploitation of covalent binding. Stability issues in peptides can be addressed via various strategies such as cyclization, incorporation of D- and non-canonical amino acids, and backbone modifications. A therapeutic small molecule ligand equipped with an electrophilic warhead binds covalently to nucleophilic groups of the target protein in a proximity-driven reaction. Irreversible covalent inhibition of an interaction can results in increased potency, selectivity, sustained pharmacodynamics, and could alleviate the effects of fast renal elimination. Therapeutic peptides may benefit from a covalent binding mode of action and alleviate pharmacokinetic limitations of this class of therapeutics. [0171] In some aspects, the disclosure is directed to synthetic peptides and peptidomimetics that are chemically modified. In some embodiments, the peptide or peptidomimetic that is modified is selected from TABLE 3, TABLE 4, TABLE 5, or TABLE 6. In some embodiments, the peptide or peptidomimetic that is modified is selected from SEQ ID NO: 1-36. Modifications may comprise chemical modifications for example such as warheads, protective groups, and pegylation. In some embodiments, the modification is at the N- or C-terminus of the peptide or peptidomimetic. In some embodiments, the modification is on a side chain of an amino acid in 47 IPTS/128777599.4 Attorney Docket No.: CLS-039WO the peptide or peptide. In some embodiments, the modification is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the modification is an arylation. In some embodiments, the arylation is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the modification is acetylation, formylation, propanoylation, hexanoylation, or myristoylation. In some embodiments, the modification is an amidated C-terminus. In some embodiments, the modification is a substitution of one or more L-amino acid with a D-amino acid. In some embodiments, the modification is a substitution of one or more amino acid with a methyl-amino acid. In some embodiments, the modification is a substitution of an α-amino acid with a β-amino acids. In some embodiments, the arylation is on a Cys, and the Cys is a position 3 or 8 of the synthetic peptide or peptidomimetic. Warheads [0172] In some aspects, the disclosure is directed to synthetic peptides and peptidomimetics comprising a warhead. In some embodiments, the peptide or peptidomimetic that is modified with a warhead is selected from TABLE 3, TABLE 4, TABLE 5, or TABLE 6. In some embodiments, the peptide or peptidomimetic that is modified is selected from SEQ ID NO: 1-36. In some embodiments, the warhead facilitates a covalent bond to a cognate protein after a chemical reaction. In some embodiments, the warhead is at the N- or C-terminus of the peptide or peptidomimetic. In some embodiments, the warhead is on a side chain of an amino acid in the peptide or peptide. In some embodiments, the warhead is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the warhead connected to the peptide by an arylation. In some embodiments, the arylation is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the warhead is on a Cys, and the Cys is a position 3 or 8 of the peptide or peptidomimetic. In some embodiments, the warhead is conjugated to the Cys via the Sulfur atom of the Cys. [0173] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000050_0001
48 IPTS/128777599.4 Attorney Docket No.: CLS-039WO . [0174] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000051_0001
. [0175] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000051_0002
. [0176] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000051_0003
. [0177] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula 49 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000052_0001
. [0178] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000052_0002
. [0179] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000052_0003
. [0180] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000052_0004
. 50 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0181] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000053_0001
. [0182] In some embodiments, the warhead is
Figure imgf000053_0002
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, - ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, - C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, - C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, - SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, -OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0183] In some embodiments, the warhead is 51 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000054_0001
. [0184] In some embodiments, the warhead is
Figure imgf000054_0002
. [0185] In some embodiments, the warhead is
Figure imgf000054_0003
wherein R10 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, - N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, - OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, - N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, -SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, - S(=O)2RC3, -S(=O)2ORC3, or -S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. [0186] In some embodiments, the warhead is 52 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000055_0001
wherein X is a halogen. [0187] In some embodiments, the warhead is
Figure imgf000055_0002
. [0188] In some embodiments, the warhead is selected from the group consisting of
Figure imgf000055_0003
53 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000056_0001
[0189] In some embodiments, the warhead is selected from the group of a sulfonyl fluoride, a phenyl carbamate, and a squaramate. In some embodiments, the warhead is conjugated to a Cys via the Sulfur atom of the Cys. Exemplary Cys substituted peptides and peptidomimetic sequences with a warhead are listed in TABLE 7. In some embodiments, the Cys substituted peptide with a warhead is selected from the group of SEQ ID NOs: 37-46. [0190] TABLE 7 lists exemplary amino acid sequences of peptide and peptidomimetics with a warhead disclosed herein. TABLE 7
Figure imgf000056_0002
54 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0191] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000057_0001
. [0192] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000057_0002
. [0193] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000057_0003
. 55 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0194] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000058_0001
. [0195] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000058_0002
. [0196] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000058_0003
. 56 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0197] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000059_0001
. [0198] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000059_0002
. [0199] In some embodiments, the peptide or peptidomimetic with a warhead has the following formula
Figure imgf000059_0003
57 IPTS/128777599.4 Attorney Docket No.: CLS-039WO IV. Complexes [0200] In some aspects, the disclosure is directed to synthetic peptides and peptidomimetics that are bound in a complex with HLA-E/β2m to form peptide/HLA-E/β2m. In some embodiments, the peptide or peptidomimetic that is complexed with HLA-E/β2m is selected from TABLE 3, TABLE 4, TABLE 5, TABLE 6, or TABLE 7. In some embodiments, the peptide or peptidomimetic that is complexed with HLA-E/β2m is selected from SEQ ID NOs: 1-47. [0201] In some embodiments, the synthetic peptides and peptidomimetics are designed to bind HLA-E/β2m. In some embodiments, the synthetic peptides and peptidomimetics bind HLA- E/β2m with low, medium, or high affinity. In some embodiments, the synthetic peptides and peptidomimetics bind HLA-E with higher affinity than VL9. In some embodiments, the peptides and peptidomimetics are covalently bound to HLA-E/β2m. In some embodiments, the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln-72, Asn-77, Thr-80, Tyr-84, Trp-97, His- 99, Glu-114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser-147, Glu-152, His-155, Gln-156, Tyr- 159, Thr-163, Cys-164, Trp-167, or Tyr-171 of human HLA-E. In some embodiments, the covalent bond is between the synthetic peptides and peptidomimetics and HLA-E residues Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E. [0202] In some embodiments, the peptide/HLA-E/β2m is modulated in binding of CD94/NKG2A. In some embodiments, the peptide/HLA-E/β2m is inhibited in binding or engaging of CD94/NKG2A or prevents activation of CD94/NKG2A. In some embodiments, the peptide/HLA-E/β2m is located on a cell. In some embodiments, the peptide/HLA-E/β2m is soluble. In some embodiments, the cell is a cancer cell. In some embodiments, the CD94/NKG2A is located on a NK cell or a T cell. In some embodiments, the inhibition of binding or engaging of peptide/HLA-E/β2m complex to CD94/NKG2A on a NK cell or a T cell modulates activity of the NK cell or the T cell. V. Preparation of Peptides [0203] Methods for producing synthetic peptide or peptidomimetic of the disclosure are known in the art such as solid phase peptide synthesis (SPPS), Fmoc-based synthesis, and Boc-based synthesis by an automatic peptide synthesizer. For example, peptides can be chemically synthesized using the sequence information provided herein and using peptide synthesis methods known in the art. The produced synthetic peptide or peptidomimetic can be modified during or after peptide synthesis with several modifications, for example with a warhead, a protective group, or pegylation. Alternatively or additionally, the peptide or peptidomimetic may be 58 IPTS/128777599.4 Attorney Docket No.: CLS-039WO modified at its amino terminus or carboxy terminus or protected by various organic groups for protecting the peptide from protein-cleaving enzymes in vivo while increasing its stability. The produced synthetic peptide or peptidomimetic can then be purified further. Purification strategies for peptides or peptidomimetics are known in the art, and include FPLC and HPLC based methods. VI. Nucleic Acids, Vectors, and Host Cells [0204] In yet another aspect, the present disclosure provides an isolated nucleic acid encoding a synthetic peptide or peptidomimetic as disclosed herein or fragment thereof. In some embodiments, the present disclosure provides one or more vectors encoding an isolated nucleic acid as disclosed herein. In some embodiments, the peptides or peptidomimetics are contemplated as recombinant peptides. In some embodiments, the present disclosure provides one or more host cells comprising a vector or isolated nucleic acid as disclosed herein. [0205] Methods for engineering host cells for peptide synthesis are known to those of ordinary skill in the art. Non-limiting examples of host cells include Bacterial Cells (e.g., Escherichia coli), Yeast Cells (e.g., Saccharomyces cerevisiae), and Mammalian Cells (e.g., CHO (Chinese Hamster Ovary), HEK293 (Human Embryonic Kidney) cells, etc.). VII. Pharmaceutical Compositions [0206] For therapeutic use, a synthetic peptide or peptidomimetic disclosed herein preferably is combined with a pharmaceutically acceptable carrier and/or an excipient. The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0207] The term “pharmaceutically acceptable carrier” as used herein refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. 59 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Co., Easton, PA [1975]. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. [0208] Pharmaceutical compositions containing a synthetic peptide or peptidomimetic disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration, e.g., oral administration. The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend upon the intended mode of administration and therapeutic application. [0209] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile solutions, the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. [0210] The term “pharmaceutically acceptable excipient” refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the invention are any of those that are well known in the art of pharmaceutical formulation and include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum 60 IPTS/128777599.4 Attorney Docket No.: CLS-039WO albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. VIII. Methods of Use [0211] The synthetic peptides and peptidomimetics of the disclosure can be used in a variety of in vitro and in vivo methods, as research reagents, for diagnostic purposes, and for therapeutic uses, based on the binding specificity of the synthetic peptides and peptidomimetics to HLA-E and on the effect on HLA-E functions of the peptides and peptidomimetics. [0212] In some embodiments, the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in a therapy (e.g., cancer). In some embodiments, the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in in the preparation of a medicament. In some embodiments, a method of treating a disease or condition (e.g., cancer), comprising administering a therapeutically effective amount of a synthetic peptide or peptidomimetic as disclosed herein. In some embodiments, the present disclosure provides a use of a synthetic peptide or peptidomimetic as disclosed herein in the preparation of a medicament for directly and/or indirectly enhancing tumor cell death, the method comprising exposing a tumor (or one or more tumor cells) and natural killer cells to the synthetic peptide or peptidomimetic. [0213] In some embodiments, the cancer or tumor cells are HLA-E-expressing. [0214] Approximately half of peripheral NK cells display the CD94/NKG2A receptor and these cells are mostly present in the CD56high fraction, which contains the more immature cells. Intratumoral NK cells have somewhat higher frequencies of CD94/NKG2A. CD94/NKG2A is also expressed on intratumoral CD8+ T cells that often display a late effector memory phenotype. The inhibitory signals induced by NKG2A receptor engagement with peptide/HLA-E results in decreased capacity of NK cells and CD8+ T cells to lyse target cells. It is contemplated, that disrupting the HLA-E- CD94/NKG2A axis may lead to reversal of the inhibitory effect that leads to immune tolerance in these cells. [0215] The synthetic peptides and peptidomimetics disclosed herein are designed to be recognized and bound covalently or non-covalently by HLA-E/β2m complexes. In some 61 IPTS/128777599.4 Attorney Docket No.: CLS-039WO embodiments, the synthetic peptides and peptidomimetics can be used to modulate HLA-E/β2m function. In some embodiments, the synthetic peptides and peptidomimetics in complex with HLA-E modulate HLA-E engagement with the CD94/NKG2A receptor heterodimer on NK or T cells. In some embodiments, the synthetic peptides and peptidomimetics in complex with HLA- E block or inhibit HLA-E engagement with the CD94/NKG2A receptor heterodimer on NK or T cells. In some embodiments, the HLA-E/β2m complex is presented on the surface of a cancer cell and the peptide or peptidomimetic binds to the HLA-E/β2m complex in a manner that blocks the HLA-E/β2m complex from engaging with the CD94/NKG2A receptor on NK or T cells. [0216] Methods for testing for peptide/HLA-E/β2m -CD94/NKG2A engagement and subsequent cell signaling are known in the art, for example by FACS, cytotoxicity assays, and cytokine release assays. IX. Kits [0217] In some embodiments, any of the synthetic peptides or peptidomimetics disclosed herein disclosed herein is assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing any of the systems or vectors disclosed herein and instructions for use. [0218] The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration. EXAMPLES 62 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0219] Below are examples of specific embodiments for carrying out what is disclosed herein. The examples are offered for illustrative purposes only and are not intended to limit scope. [0220] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, PROTEINS: STRUCTURES AND MOLECULAR PROPERTIES (W.H. Freeman and Company, 1993); A.L. Lehninger, BIOCHEMISTRY (Worth Publishers, Inc., current addition); Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL (2nd Edition, 1989); METHODS IN ENZYMOLOGY (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg ADVANCED ORGANIC CHEMISTRY 3rd Ed. (Plenum Press) Vols A and B (l992). [0221] Unless otherwise stated, all reagents and chemicals were obtained from commercial sources and used without further purification. EXAMPLE 1 - Peptide Library Design and Synthesis [0222] This example describes the design of a peptide library based on the HLA-E signal peptide VL9 VMAPRT(L/V)(V/L/I/F)L (SEQ ID NO:1 ) (VMAPRTLVL, SEQ ID NO:2 ) and subsequent library synthesis of a library of 200 million peptides synthesized by split-and-pool synthesis. [0223] Briefly, the interaction between HLA-E and VL9 with CD94/NKG2A was analyzed in an HLA-E/VL9/CD94/NKG2A complex crystal structure (the crystal structure is represented in PDB ID 3cii). Without being bound by particular theory, in the crystal structure, VL9 binds the groove formed by two α-helices of HLA-E and several amino acids within VL9 were found useful for anchoring to HLA-E (e.g., Met2 and Leu9, FIG. 1B) and binding to the receptor dimer (e.g., Arg5, Phe8). In some embodiments, Arg-5, Thr-6, and Phe-8 of VL9 were shown to be key residues involved in molecular recognition by CD94–NKG2A. The library also introduced orthogonal functional groups to replace interacting residues Arg-5, Thr-6 and Phe-8. To discover peptides that occupy the binding groove of VL9 in HLA-E but do not enable binding of the CD94–NKG2A receptor dimer, a focused library of 9-mer peptides with the formula X1-Met2- X3-X4-X5-Ala6-X7-X8-Leu9 (SEQ ID NO:3) and 10-mer peptides X1-Met2-X3-X4-X5-Ala6-X7- X8-Leu9-Lys10 (SEQ ID NO:4) (wherein each of X1, X3, X4, and X7 are independently selected from Gly, Ala, Met, Pro, Cpa, Cha, Ser, Asn, Gln, Msn, Phe, Tyr, His, Trp, 4Py, 4Af, Tha, Dff, Asp, Glu, Lys, Arg, hAr, or Aad (selected from 24 amino acids with various canonical and non- 63 IPTS/128777599.4 Attorney Docket No.: CLS-039WO canonical side chains); X5 is Gly, Ala, Val, Leu, Met, Pro, Cpa, Cba, Cha, Aoa, Phe, Trp, Mff, Dff, Tff, Tha, Nal, hPh, Dmf, Php, or Amb (selected from 21 aliphatic or aromatic amino acids); and X8 is Ser, Thr, Asn, Gln, Msn, Hyp, Asp, Glu, Lys, Arg, Dab, Orn, Aad, or hAr (selected from 14 charged or polar amino acids)). Half of the library had an extra amino acid (a Lys, Lys10) at the C-terminus at position 10 (FIGs. 1C and 1D). Canonical and non-canonical amino acids used in the peptide library are listed in TABLE 1, TABLE 2, and shown in FIG. 1D. The anchor residues Met2 and Leu9 were set constant in the library design, to enable binding to HLA-E. Substitution of Thr6 with Ala was previously shown to reduce binding to CD94/NKG2A. According to the library design, Arg5 was substituted with aliphatic and aromatic amino acids, and Phe8 was replaced by polar and charged amino acids to antagonize binding to CD94/NKG2A. C-terminal Lys was installed on half of the library to increase sequencing confidence by augmented signal intensity of fragments in secondary mass spectra. To increase the chemical diversity of the peptide collection, the 21 non-canonical amino acids were included in the library design. [0224] A focused library of ~2 × 1089- and 10-mer peptides was synthesized by split-and-pool synthesis (SPPS) on monosized resin. The 200 million-member library was used in affinity selections with immobilized HLA-E/B2M in complex with a UV-cleavable VL9 to identify putative binders by tandem mass spectrometry and de novo peptide sequencing (FIG. 1E). [0225] Briefly, a peptide library with 200 million members was synthesized on Tentagel® M NH2 resin (30 μm bead size, 0.72 mmol, 2.79 g, 70 million beads/g). The resin was placed in a fritted syringe and swollen in N,N-dimethylformamide (DMF) for 30 min. To the resin was added 4‐(4‐Hydroxymethyl‐3‐methoxyphenoxy)butyric acid (HMPB, 7 equiv.) with 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide PF6 (HATU, 6.3 equiv.) and N,N-diisopropylethylamine (DIEA, 21 equiv.) in DMF for 30 min. The reactants were removed by washing with DMF (3x), and the resin was split into two parts to couple the C- terminal amino acids Fmoc-Leu-OH, and Fmoc-Lys(Boc)-OH for the 9-mer and 10-mer library members, respectively. The amino acids (7 equiv.) were dissolved in DMF (0.5 M), N,N’- diisopropylcarbodiimide (DIC, 5 equiv.) was added, and the solution was added to the resin after 2 min of activation. 4-Dimethylaminopyridine (DMAP, 0.1 equiv.) was added, and the reaction was left for 16 h. The Fmoc protecting group was removed with 20% piperidine in DMF (2 x 5 min). Fmoc-Leu-OH (5 equiv.) was coupled to the resin functionalized with Lys with HATU (4.5 equiv) and Dipea (15 equiv) in DMF for 15 min, and, after washing and Fmoc deprotection, the resins were combined for split and pool synthesis was continued by coupling amino acids (7 64 IPTS/128777599.4 Attorney Docket No.: CLS-039WO equiv.) by activation with HATU (6.3 equiv.) and DIEA (21 equiv.) for 15 min at RT followed by Fmoc deprotection according to the library design. After final deprotection and extensive washing by DMF and CH2Cl2, the resin was dried in a vacuum chamber overnight. Peptides were cleaved from solid support using 60 mL trifluoroacetic acid/H2O/1,2-ethanedi- thiol/triisopropylsilane (TFA/H2O/EDT/TIPS; 94 : 2.5: 2.5 : 1) for 2 h at RT. TFA was evaporated to 20% of the initial volume by applying a stream of nitrogen, and the library was precipitated by addition of ice-cold Et2O. The suspension was centrifuged (4000 rpm, 5 min, 5 °C), and the residue was subjected to two more rounds of precipitation and centrifugation. After evaporation of residual Et2O, the precipitate was dissolved in 30% MeCN in H2O (+0.1% TFA) and lyophilized. The crude library lyophilizate was dissolved in 5% MeCN in H2O (+0.1% TFA) for solid phase extraction using Supelclean™ LC-18 SPE Tubes (100 mg crude library per gram of resin bed). The purified library was lyophilized and dissolved with phosphate-buffered saline (PBS) + 10% DMF to a concentration of 4 mM (20 pM per library member) for storage as single-use aliquots of 1 mL at -80°C. EXAMPLE 2 - Protein Expression and Purification [0226] This example describes the expression, purification, and refolding of HLA-E and β2m protein from inclusion bodies in E. coli and the expression and purification of CD94/NKG2A single-chain dimer from mammalian cells. Preparation of HLA-E and B2M proteins from inclusion bodies [0227] The coding sequences for HLA-E*0103 (human, residues 22-305)(SEQ ID NO:48) with a C-terminal Avitag and β2m (human, residues 21-119) (SEQ ID NO:49) were synthesized and cloned into pET29b(+) (pET29b(+)-HLAE*(hu)(22-305)-Avitag and pET29b(+)-(β2m (h)(21- 119)) (synthesized at Genewiz). [0228] The proteins were expressed in E. coli BL21 (DE3) at 37 °C until an OD600 of 0.7 and then induced with 1.0 mM IPTG for 3 hours at 37°C. For purification of proteins from inclusion bodies, pellets from 10 L cultures were resuspended in 200 mL of sucrose buffer (50 mM Tris pH 8.0, 1 mM EDTA and 25% sucrose), lysed with the addition of 0.2 g of lysozyme. After 10 minutes of lysis the solution was diluted with 500 mL of deoxycholate solution (20 mM Tris pH 7.5, 100 mM NaCl, 1% deoxycholic acid, and 1% Triton). The mixture was then adjusted to 5 mM MgCl2 and treated with 4 mg of DNAse (Sigma D-5025) until viscosity was reduced to that of water. Inclusion bodies were pelleted at 8K x g for 20 minutes after DNAse treatment and in between subsequent washes. Pellets were washed 3 times with Triton solution (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5 % Triton X-100) and further 3 times with Tris 65 IPTS/128777599.4 Attorney Docket No.: CLS-039WO solution (50 mM Tris pH 8.0, 100 mM NaCl, and 1 mM EDTA). Finally, pellets were resuspended in urea solution (25 mM MES pH 6.0, 8 M urea, 10 mM EDTA, and 0.1 mM DTT). Protein concentrations were determined by A280 using extinction coefficients for individual proteins. Refolding of biotinylated HLA-E complexes [0229] MHC complexes were refolded as previously described in Braud et al., Nature 1998, 391 (6669), 795-799 and Altman et al., Science 1996, 274 (5284), 94-96. All Chemicals were purchased from Sigma/Millipore and Peptides were purchased from New England Peptide.Briefly, 34.7 mg of [HLAE(hu)(22-305)]-Avitag and 23.7 mg [B2M(h)(21-119)] were refolded with 30 mg of VMAPRTLFL (VL9) (SEQ ID NO: 36) to form HLA-E+VL9, VMAP (C/5-Carboxyfluorescein)TLFL (VL9*5FAM), or VMAP(Anpp)TLFL (SEQ ID NO: 47) (UV- labile VL9, VL9UV) to form HLA-E+VL9UV by dilution into 1 L of refolding buffer (400 mM L- arginine, 100 mM Tris, pH 8.0, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione). Prior to addition, each protein was diluted with 4 mL of injection buffer (3 M guanidine HCl, 10 mM sodium acetate, and 10 mM EDTA). An additional 34.7 mg [HLAE(hu)(22-305)]-Avitag, diluted in injection buffer was added twice more at 12-hour intervals. Refolding solution was then subjected to buffer exchange by tangential flow filtration over a 30K MWCO PALL Omega TFF Cassette into 20 mM Tris pH 8.0. MHC complexes were then purified on an AKTA PURE using a 5 mL HiTrap Q FF column with a 25 CV gradient from 0 to 500 mM NaCl. Fractions were pooled corresponding to the refolded complex and dialyzed into 20 mM Tris pH 8.0. Complexes were then biotinylated with BirA Ligase (Avidity) and subjected to buffer exchange into TBS (20 mM Tris pH 7.5, 150 mM NaCl). Protein concentrations were determined using the extinction coefficient of the MHC complexes. CD94-NKG2A single-chain dimer expression and purification [0230] A single-chain dimer of CD94/NKG2A (hSCD) was generated by fusing human CD94 (K32-I179, Uniprot Q13241) via a GS(G4S)7GG linker to human NKG2A (P94-L333, Uniprot P26715) (SEQ ID NO:50). An N-terminal 8xHis tag was added for IMAC purification, and the whole construct was subcloned into a mammalian expression vector with a puromycin selection cassette. In a second construct (oaFc-hSCD)(SEQ ID NO:51), the N-terminal 8xHis tag was replaced by an AviTag™ followed by a one-armed human IgG1 Fc (L234A, L235A, L351K, T366S, P395V, F405R, Y407A, K409Y), linked to the hSCD insert with a GGG linker. Stable cell lines for both constructs were generated in HEK293F cells using puromycin selection. Cells were grown in Expi293 Expression Media (Thermo A1435101), and the protein was purified 66 IPTS/128777599.4 Attorney Docket No.: CLS-039WO from 1-2 L of conditioned media at a cell density of ~ 3x106 cells/mL. Conditioned media was collected by centrifugation at 3000 rpm for 20 min at 4 °C and filtered through a 0.2 uM filter unit (Corning 430515). For the his-tagged hSCD, the filtered conditioned media was loaded onto a 5 mL HisTrap™ FF column (Cytiva, 17-5255-01) on an Akta FLPC at 1 mL/min flow rate. The column was then washed with 10 CV of HBS (50 mM HEPES, 300 mM NaCl, pH 7.5) with 20 mM imidazole. The protein was eluted using a gradient elution of 20 mM – 1 M imidazole in HBS over 6 CV. Fractions were pooled, concentrated, and passed through a Superdex® 200 Increase 10/300 GL column (Sigma GE28-9909-44) in HBS. For oaFC-hSCD purification, the filtered conditioned media was loaded on a 5 mL HiTrap™ Protein G HP (Cytiva, 17-0404-01) on an Akta FPLC at 1mL/min flow rate. The column was then washed with 10 CV of PBS (Corning 21-040-CV). The protein was eluted using 0.1 M acetic acid (pH 2.7) and immediately neutralized with 1:10 the volume of 1 M Tris pH 8 and 1 M NaCl. Peak fractions were pooled, concentrated, and injected onto a Superdex® 200 Increase 10/300 GL column (Sigma GE28- 9909-44) equilibrated in PBS running at 0.5 mL/min. The main peak was collected and further concentrated for downstream applications. The final protein concentration was quantified using Pierce™ 660 nm Protein Assay Reagent (Thermo 22660) before the protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C until use. EXAMPLE 3 - Peptide Selection by Binding to HLA-E [0231] This example describes the selection of HLA-E peptide binders from the focused library by nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS). Since the HLA- E/B2M complex requires a peptide ligand for stability, the biotinylated HLA-E/B2M complex was first charged with a UV-cleavable analog of VL937 (abbreviated, e.g., as HLA- E/B2M/VL9UV). [0232] Affinity selections with HLA-E bound to UV-labile VL9 ([HLA-E+VL9UV]; [HLAE(hu)(22-305)]-Avitag + BM(h)(21-119)] + VMAP(Anpp)TLFL]*Biotin) were performed following adapted procedures for discovery of peptides from ultra-large peptide libraries described by Quartararo et al. Nature Communications 2020, 11 (1), 3183. [0233] Briefly, purified biotinylated HLA-E–B2M complex as described in EXAMPLE 2 was pre-charged with a UV-cleavable peptide resulting in HLA-E+VL9UV. Then affinity selections against [HLA-E+VL9UV] immobilized on magnetic beads were performed with the focused library from EXAMPLE 1 at a concentration of 10 pM per member on a 1 mL scale (10 fmol/peptide). MyOne Streptavidin T1 DynaBeads (10 mg/mL; 1 mg; 0.13 nmol protein binding capacity, 1 equiv.) were functionalized with biotinylated [HLA-E+VL9UV] or off-target control 67 IPTS/128777599.4 Attorney Docket No.: CLS-039WO protein (0.156 nmol, 1.2 equiv.) in wash buffer (PBS (+ 10% FCS, +0.02% Tween 20)) in a 1.7 mL microcentrifuge tube on a nutating mixer for 30 min at 4 °C. For washing, the beads were subjected to three cycles of suspending in 1 mL wash buffer followed by separation enabled by a magnetic rack. The washed beads were suspended in PBS (+ 10% FCS), and the library was added a concentration of 10 pM/member in 1.7 mL centrifuge tubes. Selections were performed under UV irradiation to cleave UV-labile VL9 and liberate the binding groove of HLA-E in presence of the peptide library. The tubes were placed on a nutating mixer and irradiated by a handheld UV lamp in 3 cm distance (λ = 366 nm) for 1 h at 4 °C. Following an incubation period, the magnetic beads bearing the binder–target complexes were removed from the mixture and washed to remove non-binding sequences. After incubation, the solution was removed on the separating rack, and the beads were subjected to three cycles of wash with PBS and separation enabled by the magnetic rack. Finally, the beads were treated with 6 M guanidine in 0.2 M phosphate buffer (pH 6.8) to denaturate the proteins and elute bound peptides. The samples were desalted by a C18 ZipTip prior to lyophilization, and dissolved in 100 mM guanidine in H2O (+ 0.1% formic acid) for analysis by nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS) on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer. nLC-MS/MS [0234] Briefly, samples from affinity selections were analyzed on a Thermo Fisher Orbitrap Fusion Lumos Tribrid Mass Spectrometer with an EASY-Spray source using a Thermo Fisher EASY-nLC 1200 System and AcclaimTM PepMapTM 100 C18 trap columns (20 mm x 75 μm, 3 μm particle size, 100 Å pore size, PN164946) and AcclaimTM PepMapTM RSLC C18 HPLC columns (150 mm x 50 μm, 2 μm particle size, 100 Å pore size, PN ES901). LC was performed with 0.1% formic acid (FA) in water (solvent A) and 80% MeCN with 0.1% formic acid in water (solvent B) prepared with LiChrosolv® water and MeCN suitable for MS from Millipore Sigma and OptimaTM LC/MS grade formic acid from Thermo Fisher Scientific. Chromatography was performed at 40 °С, with a flow rate of 300 nL/min using either of the following gradient: 1% B to 45% B (0–100 min), 45% B to 90% B (100–102 min), 90% B (102–100 min) or 1% B to 51% B (0–120 min), 51% B to 90% B (120–130 min), 90% B (130–140 min). 5 minutes after start of the gradient, MS/MS were recorded in a data-dependent method. Full MS cycle time = 3 s. Detector Type = Orbitrap. Resolution = 120000. Mass Range = Normal. Quadrupole Isolation = True. Scan Range (m/z) = 200–1400. RF Lens (%) = 30. AGC Target = 250%. Maximum Injection Time = Auto. The following filters were applied for precursor selection: Monoisotopic Precursor Selection = Peptides. Precursor Selection Range (m/z) = 200–1400. Intensity 68 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Threshold = 4.0e4. Charge States = 2–10. Dynamic Exclusion (exclusion after 1n for 30 s, mass tolerance = 10 ppm). Fragmentation was induced by collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation with higher- energy collision (EThcD). Specifications CID: Isolation Mode = Quadrupole. Isolation Window (m/z)= 1.3. Isolation Offset = off. CID Collision Energy (%) = 30, 10 ms Activation Time. Activation Q = 0.25. Detection = Orbitrap. Orbitrap Resolution = 30000. Mass Range = Normal. Scan Range Mode = Auto. AGC Target = Standard. Maximum Injection Time = Auto. 1 Microscan, Centroid Data, no Internal Calibration. Specifications HCD: Isolation Mode = Quadrupole. Isolation Window (m/z) = 1.3. Isolation Offset = Off. Collision energy (%) = 25. Detection = Orbitrap. Resolution = 30000. Mass Range = Normal. 1 Microscan, Centroid Data, no Internal Calibration. Specifications EThcD: Isolation Mode = Quadrupole. Isolation Window (m/z) = 1.3. Isolation Offset = Off. Use Calibrated Charge-Dependent ETD Parameters = True. ETD Supplemental Activation = EThcD. SA Collision Energy = 25 %. Detection = Orbitrap. Orbitrap Resolution = 30000. Mass Range = Normal. Scan Range Mode = Auto. AGC Target = Standard. Maximum Injection Time = Auto. 1 Microscan, Centroid Data, no Internal Calibration. ThermoFisher Xcalibur software package and PEAKS Studio 8.5 were used for data analysis. [0235] Selections with [HLA-E+VL9UV] and the control protein were performed in triplicates. De novo sequencing and hit identification [0236] PEAKS Studio (V8.5, Bioinformatics Solutions) was used to process raw nLC-MS/MS data and perform de novo peptide sequencing. Automated de novo sequencing was performed with a 15 ppm parent mass error tolerance, and a 0.02 Da fragment mass error tolerance. The following variable post-translation modifications were defined to resolve peptides with non- canonical amino acids: Cpa = Val +12.00; Cba = Val +26.02; Cha = Phe =6.05; 4Py = Phe +1.00; 4Af = Phe +15.01; Tha = His +16.96; Mff = Phe +17.99; Dff = Phe +35.98; Tff = Phe +53.97; Msn = Met +31.99; hAr = Arg +14.02; Aad = Glu +14.02; Aoa = Leu +28.03; Nal = Phe +50.02; hPh = Phe +14.02; Amb = Gly +76.03; Dmf = Phe +60.02; Php = Pro +90.05; Orn = Val +11.07; Hyp = Pro +15.99; Dab = Gly +43.04; Met(oxide) = Met +15.99. Up to 20 candidates were reported per scan. Data cleaning and hit identification was performed as previously described in Vinogradov et al., ACS Combinatorial Science 2017, 19 (11), 694-701. [0237] After de novo sequencing of the fragmented ions and data filtering, five nonameric peptides matching the library design were identified with high average local confidence (ALC) scores >95% and displayed selective binding to HLA-E (FIG. 2G). The sequences of the five 9- 69 IPTS/128777599.4 Attorney Docket No.: CLS-039WO mer peptides, B1, B2, B3, B4, and B5 are listed in TABLE 8. Exemplary formulas for the peptides B1-B5 are shown in FIGs. 2A-2E. TABLE 8. First generation sequences.
Figure imgf000072_0001
[0238] No 10-mer peptide was discovered with selective binding, indicating that the C-terminal Lys extension might be unfavorable for binding HLA-E. Hit sequences B1–B5 were synthesized individually by microscale SPPS to validate their inhibitory potential on the binding of CD94– NKG2A by bio-layer interferometry (BLI) and fluorescence polarization (FP) assays. [0239] Additional peptides identified in the selective binding assay are shown in TABLE B. TABLE B
Figure imgf000072_0002
70 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000073_0001
a = Cpa, b = Cba, c = Cha, d = 4Py, e = 4Af, f = Tha, g = Mff, h = Dff, i = Tff, j = Msn, k = hAr, l = Aad, m = Aoa, Nal, o = hPh, p = Amb, q = Dmf, r = Php, s = Orn, t = Hyp, u = Dab, v = Met(O), canonical amino acids are represented by their one-letter code. [0240] EXAMPLE 4A - Inhibition of CD94–NKG2A Binding to HLA-E by Peptides [0241] This example describes the analysis of inhibition of CD94–NKG2A binding to HLA-E by the peptides B1–B5 by bio-layer interferometry (BLI). Synthesis of Peptides [0242] Peptides B1-B5 identified in EXAMPLE 3 and a series of peptides with single substitution of anchor residues Met2 or Leu9 of B5 (B9, B10, B6, B7, and B8, shown in TABLE 9 and FIGs. 3A-3F) and 2 scrambled control peptides (B5 Scrambled and B9 scrambled) were synthesized. TABLE 9. B5 derived amino acid sequences
Figure imgf000073_0002
[0243] Briefly, peptides were synthesized by SPPS on a 0.05 mmol scale with a HMPB ChemMatrix resin in fritted syringes. The C-terminal amino acid (7 equiv.) was coupled to the resin by DIC (5 equiv.) and DMAP (0.1 equiv.) for 16 h at rt. The Fmoc protecting group was 71 IPTS/128777599.4 Attorney Docket No.: CLS-039WO removed using 20% piperidine in DMF for 2 x 5 min, and the subsequent amino acids (5 equiv.) were coupled using HATU (4.5 equiv.) and DIEA (15 equiv.) in DMF for 15 min at room temperature (rt). Upon completion of the sequence and deprotection of the N-terminal amine, the resin was washed with DMF and CH2Cl2, and dried in a vacuum chamber for 16 h. Cleavage from solid support and global deprotection was achieved with TFA/H2O/EDT/TIPS (94 : 2.5 : 2.5 : 1) for 2 h at rt. The solution was concentrated to 10% of its initial volume by a stream of nitrogen, and the peptides were isolated by three cycles of precipitation by ice-cold Et2O and centrifugation. The dried, crude peptides were dissolved in 30% MeCN (+0.1 % TFA) and lyophilized. [0244] Crude peptides were dissolved in 10% MeCN in water (+0.1% TFA) and purified by reverse-phase HPLC or reverse-phase flash chromatography. The purity of fractions was determined by LC-MS, and pure fractions were pooled and lyophilized. The synthesis yield (%) was calculated as pure isolated material divided by theoretical amount (based on synthesis scale) adjusted for the fraction of crude material used for purification. Exemplary yields are listed in TABLE 10. TABLE 10
Figure imgf000074_0001
72 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000075_0001
Peptide Exchange on Refolded HLA-E Complexes [0245] The biotinylated complex of HLA-E and B2M with VL9 (abbreviated as [HLA-E+VL9]) was used to evaluate the potential of de novo discovered peptides B1–B5. Ligand exchange of the peptides B1–B5 with VL9 on HLA-E was studied by incubating [HLA-E+VL9] with the competitor peptides. [0246] Briefly, 10 μM competitive peptides, VL9 peptide, or DMSO were added to 1 μM of refolded HLA-E/B2M complexes loaded with VL9 in 1X PBS (Corning 21-040-CV) and allowed to exchange for 4 h at 25 °C. HLA-E Peptide Exchange Analysis by SEC and LC-MS [0247] To determine the exchanged peptides in the HLA-E/B2M complexes, the exchanged complexes were analyzed by SEC and LC-MS. Briefly, an Agilent 1200 Series Infinity II HPLC coupled to an analytical fraction collector (Agilent, G1364F) was used to separate peptide- loaded HLA-E/B2M protein from free peptide after peptide exchange as described above. Briefly, 50 μL of peptide-exchange solution was loaded onto a Superdex 200 Increase size exclusion column (Cytiva, 28990945) at a flow rate of 0.45 mL/min in PBS at room temperature. The main UV absorbance peak at 280 nm was collected over a 200 μL volume fraction for downstream analysis. 20 μL of the collected fraction was injected into an Agilent 1200 Series Infinity II HPLC with a 2.1 x 50mm ZORBAX 80 Å Extend-C18 reverse phase column (Agilent, Part Number: 727700-902) equilibrated in water with 0.1% (v/v) formic acid and 10% (v/v) acetonitrile flowing at 0.2 mL/min with a column temperature of 40 °C. The peptide peaks were resolved using a 10 – 60 % gradient of acetonitrile in 0.1% (v/v) formic acid and eluted into a Dual Agilent Jet Steam electrospray ionization source operating at a Gas Temp of 350 °C, Drying Gas at 10 L/min, Nebulizer at 30 psig, Sheath Gas Temp at 350 °C, Sheath Gas Flow at 11 L/min, VCap voltage at 3500 V, and Nozzle Voltage at 1000 V. Peptide ions were detected with an Agilent 6230 Time-of-Flight mass spectrometer operating in positive ion mode with a Fragmentor Voltage of 150 V and Skimmer Voltage of 65 V. Spectra were analyzed using MassHunter software version B.07. Extracted ion chromatograms of base peaks associated with VL9 (VMAPRTLFL) or competitive peptides B1-B5 were integrated, and the area under the curve was used for relative quantification of peptide exchange. Exemplary results are shown in FIG. 4. Peptides B2, B3, and B5 showed over 60% ligand exchange after 4 h of incubation. B9, B10, B6, B7, and B8) showed comparable exchange with VL9. 73 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Biolayer Interferometry (BLI) [0248] The inhibitory potential of peptides B1–B5, and B9 was evaluated by measuring binding of CD94/NKG2A to HLA-E after incubation with the peptides. The peptides were were subjected to a biolayer interferometry (BLI) assay to evaluate their potential for blocking molecular recognition by CD94–NKG2A. Proteins were produced as described in EXAMPLE 2 and peptides were synthesized as described in EXAMPLE 3. In short, a biotinylated [0249] HLA-E/B2M/VL9 (VMAPRTLFL) complex was combined with a 10-fold molar excess of hit sequences in an initial step to allow ligand exchange over 16 h at ambient temperature (FIG. 14A). Biotinylated [HLA-E+VL9] (1 μM) was co-incubated with individual peptides B1- B5 (10 μM) overnight at room temperature, and the biotinylated complex was subsequently loaded on streptavidin-coated probes for analysis by BLI. (FIG. 14B). Binding inhibition of CD94–NKG2A (200 nM) to the HLA-E-loaded probes was determined relative to the parent [HLA-E+VL9] complex. Experimental controls with VL9 or DMSO during ligand exchange were included to compare binding of CD94–NKG2A to their cognate ligand. Peptides B2 and B3 showed partial inhibition of CD94–NKG2A binding to HLA-E, whereas almost no residual binding was observed for peptide B5. Peptides B1 and B4 displayed no inhibitory effect in the BLI assay (FIG. 14C). [0250] BLI was performed on a ForteBio Octet Red96e instrument. All proteins were diluted in 1X Kinetic Buffer (10X Kinetic buffer, Sartorius 181105) diluted in 1X PBS. A Blocking Buffer step was introduced to lower non-specific binding to the streptavidin (SA) tips by diluting 5% bovine serum albumin (BSA) and 20 ug/mL Biocytin (Sigma B4261) in 1X Kinetic Buffer. Refolded and biotinylated [HLA-E+VL9] complexes were loaded onto SA biosensors (Forte 18- 5019) at ~ 5 ug/ml (100nM) with the following steps: 60 sec baseline in 1X Kinetic Buffer, 180 sec loading, 60 sec blocking in Blocking Buffer and 60 sec baseline in 1X Kinetic Buffer, all at 25 °C and 1000 rpm. hSCD and oaFC-hSCD binding to immobilized HLA-E/B2M complexes was monitored at 200 nM by a 90 sec association step, followed by a 120 sec dissociation step. Data correction was performed as follows: first, aligning the data to the average of the last baseline step on the y-axis; second, by aligning the data to the dissociation step for inter-step correction; and third by filtering the data using Savitzky-Golay Filtering. B2, B3, and B5 showed 41–98%. inhibition of CD94/NKG2A binding in the biophysical assay. In particular, B5 showed only 2% of residual binding of CD94/NKG2A compared to untreated control (FIGs. 5C-5E). [0251] A series of peptides with single substitution of anchor residues Met2 or Leu9 of B5 (B9, B10, B6, B7, and B8) showed comparable inhibition of CD94/NKG2A binding (FIGs. 5C-5E). 74 IPTS/128777599.4 Attorney Docket No.: CLS-039WO A Met/Nle substitution (peptide B9) prepared to avoid the formation of oxidative side-products during synthesis, purification, and handling demonstrated comparable activity in this assay. A scrambled analog of B5 (control) showed no inhibition of binding with CD94/NKG2A [0252] Given constraints of library design, the lack of inhibitory effect of B1 and B4 could suggest that they were not successful at occupying the VL9-groove on HLA-E. To test this, peptide-stabilized HLA-E/B2M complexes were isolated after ligand exchange using size- exclusion chromatography and subsequently subjected to analysis by LC-MS to quantify the remaining fraction of VL9 in the fully-folded complexes (FIG. 14D). This revealed that peptides B2, B3, and to a larger extent B5 were able to efficiently displace the VL9 peptide as indicated by reduced abundance after exchange. In contrast, B1 only displaced minimal amounts of VL9 from the HLA-E groove while B4 did not (FIG. 14E). Peptide Inhibition of CD94-NKG2A with HLA-E/VL9/B2M [0253] For binding studies on Bio-Layer Interferometry (BLI), selected peptides were allowed to exchange with biotinylated HLA-E/B2M bound to VL9 ([HLA-E/VL9/B2M]; {HLAE(hu)(22- 305)]-Avitag + B2M(h)(21-119) + VMAPRTLFL}). In brief, lyophilized peptides were dissolved in DMSO to a concentration of 20 mM and further diluted to 20 μM in 1X PBS [21- 040-CM]. Peptide was added to biotinylated HLA-E/VL9/B2M complex at a 10:1 concentration in 1X PBS; with the final HLA-E complex concentration at 1μM and peptide concentration at 10 μM. Reactions were incubated overnight at room temperature. Exchanged complexes were diluted in 1X Octet® Kinetics Buffer (KB) [Sartorius 18-1105] in a 1:10 dilution to be run on the Sartorius Octet Red96e. Human NKG2A&CD94 Protein with an Fc tag (Acro Biosystems NC4-H5257) was diluted to 200 nM in 1X KB. Octet® High Precision Streptavidin (SAX) Biosensors [Sartorius 18-5117 ] were allowed to equilibrate in 1X KB for at least 10 min at room temperature while the plate was prepared. Blocking buffer was prepared by adding 5% BSA [Sigma A3294] and 20 ug/mL Biocytin [Sigma B4261] to 1X KB and used to quench any remaining binding sites on the SAX tips before association with the SCD. Tips were rinsed in 1X KB, HLA-E complex adsorbed onto the tips, blocked with blocking buffer, rinsed with 1X KB and then allowed to associate with SCD and then rinsed in 1X Kinetic Buffer to allow for dissociation of the SCD. The plate was allowed to shake at 1000 rpm at room temperature for the duration of the assay. [0254] The steps of the assay are as follows: Baseline 60s, Loading 180s, Blocking 60s, Baseline 60s, Association 180s, Dissociation 240s. Raw traces were observed for loading of complex and then traces were aligned to Y axis to the average of the Baseline step. The inter-step correction 75 IPTS/128777599.4 Attorney Docket No.: CLS-039WO aligned the data to the Dissociation step, and then Savitzky-Golay filtering was used to remove high-frequency noise. Data was analyzed on the Data Analysis HT software [Sartorius] and exported for Prism [Graphpad]. Fluorescence polarization (FP) assay [0255] To further understand the kinetics involved in ligand exchange, a fluorescence polarization (FP) assay was developed based on purified HLA-E/B2M complexes bound to a fluorescent analog of VL9 bearing a fluorescein at position 5 (i.e., VL9*5FAM). Hit peptides B1–B5 were added at a ten-fold molar excess, and ligand exchange was monitored at ambient temperature by a loss of FP as the VL9*FAM ligand was released from the complex (FIG. 14F). Incubation with peptides B2, B3, and B5 resulted in comparable losses of FP over 4 h at room temperature, whereas incubation with B1 and B4 resulted in minor or no changes in FP, respectively (FIG. 14G). Peptides B2 and B3 bear one homoarginine (hAr) either at position 1 or 3, whereas peptide B5 features the non-natural amino acid at both positions. Without wishing to be bound by theory, it is possible that this analog of arginine, with an extended aliphatic side chain, may contribute positively to the binding to HLA-E. Notably, treatment with DMSO resulted in only a minor drop in FP over time, suggesting both that VL9*5FAM is stably bound under these conditions and also that peptides B2, B3 and B5 actively displace VL9*5FAM out of the antigen-binding groove, potentially through an intermediate step involving both peptides bound. [0256] Displacement of VL9*5FAM by peptides B1–B5 was impaired in the presence of 10% fetal bovine serum (FBS), with B5 retaining the highest displacement efficiency under these conditions. Peptide exchange at 37°C both with and without 10% FBS revealed considerable thermal release of VL9*5FAM from its groove, even by treatment with DMSO, partially but not fully masking the competition and displacement of VL9 by peptides B1–B5. Peptide Exchange with HLA-E/VL9/B2M [0257] For Fluorescence Polarization (FP) assays, HLA-E/VL9*5FAM/B2M complex was diluted in 1X PBS (with additive when indicated) and added to a black-walled 384-well plate (Corning 3575), avoiding the edges of the plate and empty wells filled with 1X PBS to compensate for evaporation. Peptides were diluted in sterile water and added to the HLA- E/VL9*5FAM/B2M complexes at 1:20 the volume of the reaction. The reaction was read in kinetic mode on a Clariostar plate reader (Ex: 482-16; Dichroic: LP504; Em: 530-40) with shaking at 300 rpm before each reading and 10 min time points over 4 hours. A DMSO control was used to adjust the gains of channel A and B and the mP set to 200. The loss of mP was 76 IPTS/128777599.4 Attorney Docket No.: CLS-039WO measured over approximately 4 h at room temperature and 37°C. Data was analyzed in Prism and normalized by baseline subtraction of the first time point. [0258] Orthogonally, fluorescently labeled candidate peptides and controls were prepared to monitor exchange. Therein, analogs of candidate peptides and controls bearing a Cys at position 5 were subjected to conjugation with fluorescein-5-maleimide (Supporting Information, Section 3). HLA-E/B2M/VL9 was diluted in 1X PBS (with additive when indicated) and then added to a black-walled 384-well plate (Corning 3575), avoiding the edges of the plate and empty wells filled with 1X PBS to compensate for evaporation. Peptides were diluted in sterile water and added to the HLA-E/B2M/VL9 complexes at 1:20 of the volume of the reaction. The reaction was read in kinetic mode on a Clariostar plate reader (Ex: 482-16; Dichroic: LP504; Em: 530- 40) with shaking at 300 rpm before each reading and 10 min time points over 4 hours. A DMSO control was used to adjust the gains of channel A and B and the mP set to 200. The loss of mP was measured over approximately 4 h at room temperature and 37°C. Data was analyzed in Prism and normalized by baseline subtraction of the first time point. VL9 Abundance After Exchange (LC-MS) [0259] HLA-E/VL9 complex after incubation with selected peptides for exchange was injected through an Agilent Infinity II HPLC system onto a Superdex 200 Increase 5/150GL size exclusion chromatography column (Cytiva 28990945) and fractions of the HLA-E/Peptide complex were collected to isolate peptides bound onto HLA-E away from free unbound peptides. The HLA-E/Peptide complex was then injected onto another Agilent Infinity II HPLC system onto a MabPac RP, 4uM, 2.1X50mm reverse phase column (ThermoFisher 088648) in tandem with an Agilent 6230 Time-Of-Flight mass spectrometer equipped with Dual Agilent Jet Stream electrospray ionization source operating in positive ion mode. Peptide and HLA-E were resolved on the column over a 10-70% Acetonitrile in 0.1% (v/v) formic acid gradient over 10 minutes at 0.3 mL/min with a column temperature of 70°C and the base ion peak of VL9 was integrated by the extracted ion chromatogram function using MassHunter B.07 software to measure the abundance of remaining VL9 bound onto HLA-E post exchange with our synthetic peptides. EXAMPLE 4B – Substitution of Met2 and Leu9 in peptide 5 with non-canonical amino acids [0260] As peptide B5 displayed near complete exchange with VL9 and had the maximal inhibitory effect on binding to CD94–NKG2A, we explored the substitution of its anchor residues Met-2 and Leu-9 with non-natural amino acids (FIG. 5B). These residues were fixed 77 IPTS/128777599.4 Attorney Docket No.: CLS-039WO positions in the initial library design as they had been identified as possible key anchor sites for binding to HLA-E7. However, to explore conservative substitutions to enhance the binding properties and chemical stability of the peptides substitutions were tested. Peptides B6, B7, and B8 were synthesized as analogs of peptide B5 featuring non-canonical norleucine (Nle), 8- aminooctanoic acid (Aoa), and ß-cyclohexyl alanine (Cha), respectively, to explore alternative hydrophobic residues at anchor position 9 (FIG. 5A). At position 2, there was substitution of the oxidation-sensitive thioether in Met with non-canonical Nle and methoxinine (Mox) in peptides B9 and B10, respectively, in order to prevent the formation of the oxidation side product in downstream applications (Grob, N. M.et al. (2017) Journal of Peptide Science). [0261] Monitoring ligand exchange by size-exclusion chromatography followed by LC-MS confirmed that peptides B6–B10 were capable of displacing VL9 from the antigen-binding groove of HLA-E to a high degree (FIG. 5F). Moreover, all analogs of peptide B5 displayed potent inhibition of molecular recognition by CD94–NKG2A in BLI assays, which was marginally reduced for peptide B10 in this series (FIG. 5G). A high degree of ligand exchange monitored by loss of fluorescence polarization was also observed with peptides B6–B10, indicating successful competition with VL9*5FAM (FIG. 5H). A sequence isomer of peptide B9 with scrambled residues (B9 scrambled), showed no inhibitory activity in the BLI assays and no displacement of VL9*5FAM in FP assays, indicating that proper sequence assembly is crucial for ligand exchange and hence PPI inhibition. [0262] As with the B1-B5 series, displacement of VL9*5FAM by peptides B6-B10 was impaired in the presence of 10% FBS, with peptides B8 and B9 retaining the highest displacement efficiency under these conditions (data not shown). As previously observed, peptide exchange at 37°C resulted in considerable thermal release of VL9*5FAM from the antigen-binding groove of HLA-E with control treatment with DMSO, resulting in only marginally lower loss of FP through treatment with B6–B10 (data not shown). Of all the series, only peptides B8 and B9 retained some excess ability to displace VL9*5FAM in complex media at 37 °C. EXAMPLE 5 - Covalent Binding of VL9 Analogs to HLA-E [0263] This example describes the introduction of electrophilic warheads into VL9 derived peptides and the covalent binding of the armed peptides to HLA-E. [0264] Covalent inhibition is a useful strategy to increase potency, selectivity, and pharmacodynamics of drugs, and alleviates the effects of fast renal elimination of peptides. Several residues in the proximity of VL9 in the binding groove of HLA-E, e.g., Tyr-7, Lys-146, 78 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Tyr-159, Tyr-171, bear nucleophilic groups potentially amenable for covalent binding through a Sulfur(VI) Fluoride Exchange (SuFEx) electrophile contained by the meta-substituted aryl sulfonyl fluoride (mSF) warhead (FIG. 6A). [0265] To determine the optimal site on an HLA-E-binding peptide for efficient cross-linking with the target protein, an electrophile scan with VL9 the endogenous ligand of HLA-E was performed. A library of peptide variants of VL9 with single Cys mutations were synthesized for every position in the sequence. Additionally, peptide B9 (described in EXAMPLE 4) was equipped with an electrophilic warhead to further increase its potential as inhibitor of the HLA- E/CD94–NKG2A interaction. Position 8 of the B9 peptide (B9_8*) was selected for the installation of the aryl sulfonyl fluoride warhead, as this position led to the highest conversion in the electrophile scan with VL9. The sequences of the Cys substituted peptides VL9-1 Cys(mSF) to VL9-9 Cys(mSF) (also denoted as VL9-1* to VL9-9*) and B9_8 Cys(mSF) (also denoted as B9_8*) with the warhead position marked as mSF are shown in TABLE 11. TABLE 11
Figure imgf000081_0001
Synthesis of Palladium Oxidative Addition Complex, (RuPhos)Pd(m- benzenefluorosulfonyl)Br, 1 [0266] To a 1 dram vial equipped with a magnetic stirbar was added 3-bromobenzenesulfonyl fluoride (33 mg, 0.14 mmol, 1.1 equiv) and RuPhos (65 mg, 0.14 mmol, 1.1 equiv). The vial was loosely sealed with a screw cap and brought into a nitrogen-filled glovebox. Cyclohexane (1.5 mL) and (cod)Pd(CH2TMS)2 (50 mg, 0.13 mmol, 1.0 equiv) were added in that order, resulting in a clear solution. The reaction vessel was sealed tightly, removed from the glovebox and allowed to stir at room temperature overnight. The reaction mixture was opened to atmosphere, pentane (1.5 mL) was added, and the mixture allowed to stand in at –20 °C for 2 h. The resulting precipitate was collected by vacuum filtration and washed twice with a minimal amount ice- 79 IPTS/128777599.4 Attorney Docket No.: CLS-039WO cooled pentane. Drying under high vacuum afforded the desired product as a grey solid (38 mg, 33% yield), which was used without further purification. An exemplary synthesis diagram is shown in FIG. 7. The identity and integrity of the Palladium Oxidative Addition Complex was analyzed with 1H NMR, 13C NMR, 31P NMR, and 19F NMR.
Figure imgf000082_0001
– 7.57 (m, 4H), 7.55 – 7.44 (m, 2H), 7.44 – 7.33 (m, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.89 (ddd, J = 7.7, 3.1, 1.3 Hz, 1H), 6.68 (d, J = 8.5 Hz, 2H), 4.64 (hept, J = 6.0 Hz, 2H), 2.15 – 2.02 (m, 2H), 1.89 – 1.48 (m, 13H), 1.38 (d, J = 5.8 Hz, 6H), 1.27 – 1.06 (m, 5H), 1.02 (s, 6H), 0.84 (s, 1H), 0.58 (s, 1H) ppm. 13C NMR (101 MHz, CD2Cl2) δ 146.4, 146.3, 145.2145.0, 140.7, 136.10, 136.06, 135.8, 133.3, 133.2, 133.1, 132.7, 131.52, 131.49, 131.47, 130.77, 130.75, 127.19, 127.13, 127.0, 123.55, 111.0, 108.2, 71.9, 28.95, 28.90, 28.82, 27.48, 27.32, 27.29, 27.22, 26.5, 22.4, 21.8. (Observed complexity due to C–F and C–P coupling) ppm. 31P NMR (162 MHz, CD2Cl2) δ 31.72 ppm 19F NMR (377 MHz, CDCl3) δ 66.54 ppm.) FT-IR (Diamond-ATR, neat) ῦmax 2973.46 (w), 2923.73 (m), 2853.05 (w), 1455.98 (m), 1400.82 (s), 1204.79 (s), 1112.97 (m). HRMS calcd for C36H47O4FPPdS [M-Br]+: 731.1946 Da, found: 731.1962 Da. Synthesis of Peptides with Covalent Warheads [0267] Cys-modified peptides were synthesized as outlined in EXAMPLE 4 and cleaved from the resin using TFA/phenol/H2O/ thioanisole/EDT (82.5 : 5 : 5 : 5: 2.5) for 2 h at rt and isolated by three cycles of precipitation with ice-cold diethyl ether and centrifugation. The palladium oxidative addition complex of meta-substituted aryl sulfonyl fluoride electrophilic warhead (2.25 equiv.) was dissolved in MeCN and added to crude peptides (1 equiv.) dissolved in HEPES (0.5 M, pH = 7.0) over 30 sec. The solution was mixed thoroughly and allowed to react for 30 min at rt. AcOH was added, and the solution was diluted with H2O. The peptide was isolated from the reaction mixture by reverse-phase flash chromatography using a Sfär Duo C18 column (12 g). Yield is expressed as % of isolated, pure peptides over crude peptides used for conjugation of the warhead. Exemplary peptide yields and calculated peptide mass are shown in TABLE 12. TABLE 12. Peptide yields and calculated peptide mass for Cys-modified peptides
Figure imgf000082_0002
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Figure imgf000083_0001
[0268] The Cys peptide variants were equipped with a mSF warhead in a Pd-mediated coupling using Pd oxidative addition complex 1 (FIG. 6A and 6B) X* indicating position functionalized with the electrophilic warhead). Electrophilic analogs of VL9 were subjected to cross-linking reactions in a 10-fold excess with [HLA-E+VL9] for 4 h at 37 °C, and the amount of covalently bound HLA-E was determined by LC-MS as described in EXAMPLE 4. Installing the electrophilic warhead at positions 1, 2, 5, 6, or 9 led to no or only trace amounts of cross-linking with HLA-E, whereas substantial cross-linking was observed with the aryl sulfonyl fluoride at positions 4 (36%), 7 (22%), and in particular at positions 3 (73%) and 8 (87%) (FIG. 6C). [0269] The electrophilic designer peptide B9_8* (FIG. 8A) but not electrophilic VL9_8*, derived from the endogenous ligand of HLA-E, was able to reduce the binding of CD94– NKG2A to HLA-E by 85% as was observed by BLI after a 2 h incubation of [HLA-E+VL9] with the electrophilic peptides (FIG. 8B). 43% of cross-linked HLA-E–B9_8* could be observed after 4 h of incubation illustrated in FIG. 6B as determined by LC-MS (FIG. 8C). B2M, which is co-expressed with HLA-E for stabilization of the MHC class I molecule and is present at equimolar concentrations in the cross-linking reaction, bears several nucleophilic residues (Lys, Cys, His, Ser, Thr, Tyr). No mass shift was observed for B2M in the cross-linking experiment, indicating that B9_8* binds and reacts specifically with HLA-E (FIG. 8C). Cross- linking reactions with HLA-A, another MHC class I molecule, showed no conversion with B9_8* within 4 h. EXAMPLE 6 – Determination of Inhibition of CD94–NKG2A Binding to HLA-E by Peptides [0270] This example describes the analysis of inhibition of CD94–NKG2A binding to HLA-E by exemplary modified peptides by bio-layer interferometry (BLI). 81 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0271] Additional VL9 based HLA-E binding peptides were designed either based on the library described in EXAMPLE 1. [0272] Further, peptides based on the ligand for the NKG2A/CD94 inhibitory receptor in mice, the nonclassical MHC molecule Qa-1b, the mouse HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. [0273] Briefly, proteins and VL9 or Qdm derived peptides were produced as described in EXAMPLE 2 and EXAMPLE 3. BLI measurements to determine inhibition were performed as described in EXAMPLE 4. [0274] Exemplary modified peptides that were used in the inhibition of CD94–NKG2A binding to HLA-E measured by measurements are shown in TABLES 13, 14, and 15. TABLE 13
Figure imgf000084_0001
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Figure imgf000085_0001
BLOD = below the limit of detection [0275] TABLE 14 shows exemplary Qdm derived peptides and BLI results. TABLE 14
Figure imgf000085_0002
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Figure imgf000086_0001
BLOD = below the limit of detection [0276] TABLE 15 shows exemplary VL9 derived peptides with additional amino acids at the N- terminus and BLI results. TABLE 15
Figure imgf000086_0002
BLOD = below the limit of detection [0277] TABLE 16 shows exemplary VL9 derived peptides. TABLE 16
Figure imgf000086_0003
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Figure imgf000087_0001
EXAMPLE 7 – Determination of Covalent Binding of VL9 modified Analogs to HLA-E [0278] This example describes the analysis of crosslinking of modified VL9 based peptides to HLA-E. [0279] Briefly, crosslinking reactions were performed as described in EXAMPLE 5. Exemplary peptides and crosslinking results are shown in TABLE 17. TABLE 17
Figure imgf000087_0002
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Figure imgf000088_0001
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Figure imgf000089_0001
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Figure imgf000090_0001
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Figure imgf000091_0001
EXAMPLE 8 – Determination of Binding of VL9 modified Analogs to HLA-E by Fluorescence Polarization [0280] This example describes the analysis of binding of modified VL9 based peptides to HLA- E by Fluorescence Polarization (FP). 89 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0281] Briefly, proteins and VL9 or Qdm derived peptides were produced as described in EXAMPLE 2 and EXAMPLE 3 and binding to HLA-E was measured with Fluorescence Polarization. [0282] Exemplary peptides and FP results are shown in TABLE 18. TABLE 18
Figure imgf000092_0001
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Figure imgf000093_0001
EXAMPLE 9 – Determination of Binding of VL9 modified Analogs to HLA-E [0283] This example describes the analysis of binding of peptides to HLA-E by Fluorescence Polarization (FP), BLI or crosslinking. [0284] Briefly, proteins and VL9 or Qdm derived peptides are produced as described in EXAMPLE 2 and EXAMPLE 3 and binding to HLA-E is measured with Fluorescence Polarization, BLI, or crosslinking as described in EXAMPLE 4, 5, or 7. [0285] Exemplary peptides that can be analyzed are shown in TABLE 19. 91 IPTS/128777599.4 Attorney Docket No.: CLS-039WO TABLE 19
Figure imgf000094_0001
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Figure imgf000095_0001
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Figure imgf000096_0001
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Figure imgf000097_0001
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Figure imgf000098_0001
EXAMPLE 10 – Structure activity relationship (SAR) studies on lead peptide B9 [0286] In this experiment, peptide B9 was selected for further evaluation on the basis of efficient displacement of VL9, maximal inhibition of binding by CD94–NKG2A, and the absence of Met at position 2, preventing potential side-products resulting from oxidation. To understand the sequence determinants of peptide B9 and its ability to stably bind and occupy the VL9-binding groove on HLA-E, the thermostability of HLA-E/B2M complexes was determined after VL9 was exchanged for with Ala-substituted analogs of B9 for 16 h at ambient temperature. As expected, the substitution of key anchor positions Nle-2 and Leu-9 with alanine revealed the least thermally stable complexes with a melting temperature shift of approximately 6°C below the fully exchanged HLA-E/B2M/B9 complex (FIG. 5J). Alanine substitutions at residues hAr-1, 96 IPTS/128777599.4 Attorney Docket No.: CLS-039WO hAr-3 and Cha-7 revealed some degree of thermal destabilization relative to the B9-loaded complex, with hAr-3 having an almost equally destabilizing effect as the anchor-site mutants. [0287] Finally, competition with DMSO alone revealed some degree of destabilization relative to the B9-loaded complex, likely mediated by thermal release of the VL9 peptide over time (FIG. 3H). This effect was recapitulated by Alanine-mutants at positions 4, 5, 6, and 8. These observations were confirmed when we analyzed VL9 abundance by LC-MS after a 16-hour exchange of a HLA-E/B2M/VL9 complex with the Ala-substituted analogs of B9, as well as binding assays with purified CD94-NKG2A complexes by BLI (data not shown). The amount of residual VL9 was highest when positions 1, 2, 3, and 9 in B9 were mutated to Alanine, corresponding to the weakest inhibition of binding by CD94–NKG2A. Surprisingly, both the hAr-1-Ala and Cha-7-Ala analogs showed near-complete inhibition of binding to CD94-NKG2A by BLI, despite incomplete displacement of VL9 and at least partial thermal destabilization of the complex. EXAMPLE 11 – Crystal structure of HLA-E bound with peptide B9 [0288] In order to gain further insight into the binding mode of peptide B9 to HLA-E and the factors contributing to inhibition of binding to CD94–NKG2A, B9 was crystallized in complex with HLA-E and B2M to solve the structure to 1.51 Å resolution (FIG. 15A). All residues of B9 were well resolved with the exception of Mff-5 that featured incomplete sidechain density, possibly due to high flexibility. By design, B9 displayed a similar mode of binding to HLA-E as VL9 at anchor positions 2 and 9 with comparable backbone interactions at position 1 forming a hydrogen bond with the hydroxyl groups of Tyr-7 and Tyr-171 on HLA-E (FIGs. 15C and 15D). However, clear differences were observed in the side chain of hAr-1 that is engaged in a triple π stacking interaction with HLA-E residues Trp-167 on the α2-helix and Arg-62 on the α1-helix leading to a conformational shift of Arg-62 as compared to its position when bound to VL9 based on alignment with the NKG2/CD94/HLA-E/B2M/VL9 structure (FIGs. 15C and 15D). hAr-1 is also stabilized by a water-mediated network of hydrogen bonds with Glu-63 and Arg-62 on the α1-helix involving also the backbone atoms of Nle-2 (FIG. 15C). Nle-2 binds in a pocket where it partakes in hydrophobic interactions with Met-45 of HLA-E in a similar manner as observed for Met-2 of VL9 (FIGs. 15C and 15D). HLA-E residue Trp-167 on the α2-helix, and Arg-62 and Gln-64 on the α1-helix form serial interactions with the side chains of hAr-1 and Gln-4 of peptide B9. Therein, Gln-4 and Mff-5 are covered by Arg-65 of HLA-E (FIG. 15C). The side chain of hAr-3 forms a salt bridge to Glu-114 on the β-sheet floor and a hydrogen bond to Gln-156 the α2-helix in HLA-E and an additional hydrogen bond to the carbonyl oxygen of 97 IPTS/128777599.4 Attorney Docket No.: CLS-039WO Mff-5 in B9 (FIG. 15C). Ala-6 in B9 is located in a pocket formed by hydrophobic interactions with Thr-70 and Ile-73 on the α1-helix of HLA-E (FIG.15C). The side chain of residue Cha-7 in B9 forms hydrophobic interactions with Trp-133 and Leu-124 on the β-sheet floor of HLA-E (FIG. 15C). The sidechain of the anchor residue Leu-9 sits in an adjacent pocket formed by Tyr- 123, Leu-124, and Tyr-118 on the β-sheet floor of HLA-E (FIG. 15C). The backbone of Leu-9 in B9 forms hydrogen bonds to Asn-77 and Tyr-84 on the α1-helix and Ser-143 on the β-sheet floor of HLA-E, in analogy to VL9, and engages in an additional hydrogen bond with Lys-146 (FIGs. 15C and 15D). [0289] In order to understand the inhibitory mechanisms of HLA-E/B2M/B9 on CD94–NKG2A interactions, the structure with the crystal structure of the complex of human CD94–NKG2A was aligned with HLA-E/B2M/VL9 (herein referred to as the coreceptor-bound structure). This analysis suggested that B9 likely interferes with coreceptor binding in line with the experimental observations. Residues 148–152 of the α2-helix of HLA-E, which are an important hub for CD94-NKG2A molecular recognition, were found disordered in the structure of the HLA- E/B2M/B9 complex but well-ordered in the coreceptor-bound complex structure (FIGs. 15E- 15F). In the coreceptor-bound structure, Glu-152 in HLA-E forms a salt bridge with Arg-5 of VL9, and Ser-151 forms a hydrogen bond to Arg-137 of NKG2A (FIG. 15F). When comparing the B9-bound structure with the coreceptor-bound structure, HLA-E residue Gln-156 clashed sterically with hAr-3 of B9 suggesting a conformational change induced by binding of B9 (FIG. 15E) as a result of the strategic substitution at position 5. It is conceivable that the observed conformational changes within residues 148–152 of HLA-E in complex with B9 may also prevent a hydrogen bond between NKG2A residue Arg-137 and HLA-E residue Ser-151 (FIGs. 15E-15F). Additionally, sterical clashes would result between the α1-helix residue Arg-62 of HLA-E and B9 residue hAr-1, and between hAr-8 of B9 residue Gln-112 of CD94 (FIGs. 15E- 15F). As Gln-112 of CD94 forms hydrogen bonds to both backbone heteroatoms of VL9 residue Thr-6 (FIG. 15F), this interaction is likely hindered in the complex with B9 (FIG. 15E). Thus, our structural insights suggest that B9 may indeed prevent molecular recognition in the absence of conformational changes by CD94–NKG2A. [0290] Crystal structures of HLA-E in complex with Mtb44 revealed that the overall backbone orientation of B9 in the HLA-E peptide-binding groove bears higher resemblance to the conformation of the Mtb44 peptide than that of VL9 (FIG. 15B). Interestingly, Mtb44 contains an arginine at the first position in a similar orientation as hArg-1 in B9, but the extended interaction network as observed with B9 residues Gln-4 and Mff-5 is not present for the 98 IPTS/128777599.4 Attorney Docket No.: CLS-039WO pathogenic peptide because it features residues with short sidechains, Ala-4 and Lys-5 respectively, at these positions (FIG. 15B). Anchor residues at position 2 (Leu in Mtb44 and Nle in B9) assume a similar orientation, and Leu-9 is featured in both peptides in the same binding position, whereas the residues in the center of the peptides differ considerably (FIG. 15B). Notably, only the structure bound with B9 shows drastic disordering of HLA-E residues 148– 152 in the α2-helix, suggesting a mechanism by which interactions with CD94/NKG2A could be impaired, that is not present in structures bound with Mtb44 (FIGs. 15B and 15E). Materials and Methods [0291] HLA-E-01:03 protein containing amino acids 22-297 was expressed, purified, and refolded with B2M and B9 peptide as described above. The complex was dialyzed into 20 mM Tris pH 8.0, 100 mM NaCl and concentrated to 13.52 mg/mL for use in crystallization. HLA- E/B2M/B9 complex crystals were grown by hanging drop vapor diffusion with a well solution consisting of 100 mM sodium HEPES/MOPS pH 7.5, 20% PEG 500 MME, 10% PEG 20,000, 30 mM sodium nitrate, 30 mM sodium phosphate dibasic, and 30 mM ammonium sulfate at 18 °C. Crystals were cryo-protected in well solution supplemented with 15% trehalose and flash frozen in liquid nitrogen for data collection. [0292] Data was collected on beamline 8.3.1 at the Advanced Light Source. Data was collected on a single crystal rotated 360° in increments of 0.2° for a total 1200 frames. Data was processed by XDS (Kabsch 2010) into P1211 space group, and scaled with aimless (Evans and Murshudov 2013) with a CC1/2 cutoff of 0.5, resulting in a final resolution of 1.51 Å. The HLA- E/B2M/VL9 peptide complex (pdb 1ktl) was modified to remove the peptide and used as a search model in Phaser (McCoy et al. 2007) to solve the structure (pdb 1ktl). Phaser fit one copy of the HLA-E/B2M complex into the asymmetric unit of the crystal. Clear electron density for B9 was observed and the peptide was built into it. The structure was manually adjusted to the electron density to add and delete regions that differed from the search model and refined with Phenix (Liebschner et al. 2019). The final structure is composed of HLA-E residues 2-146 and 153-276, B2M residues 1-100 (using the mature protein numbering after signal peptide removal), all B9 residues, one sulfate and four polyethylene glycol molecules (from the crystallization condition), one trehalose molecule (from the cryoprotectant), and 420 waters. EXAMPLE 12A – Biophysical Assay of B9 and Mtb44 [0293] Mtb44 binds to the antigen-binding groove of HLA-E with similar efficiency as VL9, and has been shown to stimulate restricted CD8+ T-cell responses in infected and vaccinated humans. The high stability of Mtb44 in complex with HLA-E/B2M has been extensively 99 IPTS/128777599.4 Attorney Docket No.: CLS-039WO evaluated. Given the conformational similarities adopted by B9 and Mtb44 in the peptide- binding groove of HLA-E (FIG. 15B), this example compared these two peptides in biophysical and cellular assays. [0294] Control VL9, B9, and Mtb44 and scrambled analogs VL9scr, B9scr, and Mtb44scr were synthesized and analyzed. Both Mtb44 and B9 efficiently displaced VL9 and VL9*5FAM from HLA-E/B2M as analyzed by LC-MS and fluorescence polarization assays (FIGs. 16A and 16B), while the scrambled analog of B9 did not. Given the redundancy in the sequence of Mtb44 (i.e. NH2-RLPAKAPLL-COOH), its scrambled analog is inevitably similar (i.e., NH2- PPALLALKR-COOH), which resulted in only partial displacement of VL9 and VL9*FAM as measured by LC-MS and fluorescence polarization assays (FIGs. 16A and 16B). [0295] In order to compare the stability of the HLA-E/B2M complex loaded with B9, VL9, and Mtb44, thermal denaturation assays were carried out after VL9-peptide exchange for 16 h at ambient temperature in the presence of each peptide, scrambled analogs, or DMSO. Both Mtb44 and the peptide B9 resulted in complexes with higher thermal stability relative to HLA- E/B2M/VL9, with B9 providing the highest degree of thermal stabilization by increasing the melting temperature almost 2°C (FIGs. 16C and 16D). In contrast, all scrambled analogs resulted in similar losses in thermal stability relative to B9 and VL9-loaded complexes as the DMSO control. The same peptide-loaded complexes were subjected to a BLI assay to evaluate their potential for blocking molecular recognition by CD94–NKG2A. In agreement with the extent of VL9 displacement observed in FIG. 16A, complexes loaded with B9 and Mtb44 resulted in nearly complete inhibition of co-receptor binding, with B9 excelling over Mtb44. HLA-E loaded with VL9 retained the ability to bind to the same extent as samples treated with DMSO and scrambled controls, with the exception of Mtb44scr, which, as expected from its capacity to partially displace VL9, resulted in partial inhibition of co-receptor binding (FIG. 16E). Given that B9 and Mtb44 behaved similarly when loaded onto the HLA-E/B2M complex, we wanted to evaluate their ability to load onto the VL9-binding groove in the presence of competing VL9. To this end, competition peptide exchange assays were carried out, in which VL9-loaded complexes were incubated with a 10-fold molar excess of peptide mixtures for 16 h at ambient temperature. In these assays, the total peptide concentration remained constant while the relative concentrations of B9 or Mtb44 with respect to VL9 were progressively increased. Thereafter, the complexes were subjected to BLI assays to monitor CD94-NKG2A binding. These assays revealed that B9 is capable of outcompeting VL9 with a half-maximal inhibitory effect at a 1:3 molar ratio with respect to VL9, whereas Mtb44 only reached half-maximal inhibition at a 3:1 100 IPTS/128777599.4 Attorney Docket No.: CLS-039WO molar ratio with respect to VL9, suggesting that B9 is better at competing with VL9 for the binding groove on HLA-E (FIG. 16F). In order to test this, and given that the thermostability of the B9- and Mtb44-loaded HLA-E/B2M complexes was found to be comparable, the binding association rates of the peptides was investigated. To this end, a gain of FP assay was designed (FIG. 16G) in which HLA-E/B2M/VL9 complexes were incubated with a 10-fold molar excess of fluorescent analogs of the peptides tagged with fluorescein at position five (i.e., VL9*5FAM, B9*FAM, and Mtb44*FAM, and scrambled analogs). Increases in FP indicative of loading of the fluorescent peptide onto the antigen-binding groove of HLA-E was monitored. These assays revealed that B9*5FAM is the most efficient at loading, reaching steady-state FP levels 1.3-fold and 4-fold higher than Mtb44*5FAM and VL9*5FAM, respectively (FIG. 16H). None of the scrambled peptide analogs were able to bind onto HLA-E/B2M and displace VL9. Of the three peptides, VL9 is the only peptide that utilizes position five to bind HLA-E directly (FIG. 15D), which could help explain why the VL9*5FAM analog was not very efficient at loading. [0296] As observed in previous loss of FP assays, the displacement of VL9*5FAM by VL9, B9, and Mtb44 peptides was impaired by the addition of 10% FBS, with Mtb44 retaining the highest displacement efficiency under these conditions. Similarly, in the gain of FP assay, addition of 10% FBS greatly impaired the ability of B9*5FAM, Mtb44*5FAM, and VL9*5FAM to load onto the HLA-E peptide-binding groove, and ablated the advantage of B9*5FAM over Mtb44*5FAM as observed previously in serum-free assays. Surprisingly, peptide exchange at 37°C did not impair the ability of B9*5FAM to displace VL9 and occupy the HLA-E binding groove, while the ability of Mtb44*5FAM to displace VL9 and occupy the HLA-E groove was reduced by a factor of 3. [0297] Overall peptide B9 outperformed Mtb44. Collectively, these data suggest that B9 is a potent competitor of VL9 for binding and stabilization of the HLA-E/B2M complex, and shows superior properties in direct comparison with the well-characterized pathogen-derived binder Mtb44. Materials and Methods [0298] Thermal Stability: In order to determine the thermostability of the complex we ran NanoDSF on exchanged complexes. Peptides were diluted in sterile water and then added to HLA-E/B2M/VL9 complex (2μM) to a final concentration of 20μM in 1X PBS. Complexes were incubated overnight at room temperature and then run in duplicate. Microcapillaries (NanoTemper PR-C002) were used to draw up approximately 10μL of sample and then loaded into a Prometheus Panta. Samples were then run for thermal shift. The ratio of the first derivative 101 IPTS/128777599.4 Attorney Docket No.: CLS-039WO was calculated using the Panta Software and the data exported and analyzed on Prism to plot the ratio as well as the change in thermal stability compared to controls. EXAMPLE 12B – Cellular Assays of B9 and Mtb44 [0299] Having established that B9 is a strong binder to HLA-E/B2M, capable of displacing VL9 and stabilizing HLA-E/B2M in solution, this example tested whether it can likewise do so in the context of an HLA-E/B2M complex expressed at the cell surface, and if so, whether it still can efficiently inhibit interactions with soluble and cell-surface expressed CD94–NKG2A co- receptors. [0300] First tested was if B9 can efficiently displace VL9 on HLA-E/B2M complexes at the cell surface. To this end, SCaBER cells were pre-treated with INFγ overnight, followed by a one- hour incubation in 10 μM VL9 (VMAPRTLFL), and then by a serial dilution of VL9, B9, Mtb44 and their scrambled counterparts. HLA-E levels were measured by FACS using peptide-agnostic anti-HLA-E antibodies (i.e. 3D12-APC), and interactions with CD94–NKG2A were measured with tetramerized biotinylated coreceptors using fluorescent streptavidin. As can be observed in FIGs. 17A and 17B, only VL9, B9 and Mtb44, but not their scrambled counterparts, stabilized HLA-E/B2M complexes at the cell surface, quickly saturating at or above 5 μM peptide concentration. On the contrary, of all peptides, only VL9 retained the ability to bind to tetramerized CD94–NKG2A at all concentrations tested. Coupled with similar levels of HLA-E at the cell surface, this observation indicates that both B9 and Mtb44 had efficiently occupied HLA-E’s peptide-binding-groove. [0301] Next tested was whether B9 or Mtb44 are better at displacing and/or competing with VL9 for HLA-E binding. This was done by incubating SCaBER cells in a solution with constant total peptide concentration (i.e. 20 uM), where the relative ratios of VL9 to B9, or Mtb44, progressively varied from 20:0 to 0:20, and detected cell-surface HLA-E and CD94–NKG2A interactions as described above. As shown in FIG. 17C, HLA-E levels were saturated in all conditions; however, in the B9 competition assay the rate of loss of CD94–NKG2A interactions was higher than that of Mtb44, suggesting that B9 is a more efficient competitor for VL9 than Mtb44 also in a cellular context, see FIG. 17D and FIG. 16F. [0302] Lastly, the experiment tested whether cells presenting B9 on HLA-E/B2M complexes at the cell surface have reduced interactions with CD94–NKG2A expressed on the surface of other cells. To this end, a Jurkat reporter cell line harboring a CAR composed of a CD94–NKG2A single-chain dimer linked to an NFAT-Lucia reporter system was used. In this system, binding to HLA-E/B2M expressing cells resulted in the measurable secretion of Lucia® luciferase (FIG. 102 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 17E). In order to test whether B9 can modulate cell-cell interactions through this pathway, SCaBER cells were loaded with a saturating concentration of VL9 for one hour, followed by two-hour incubation with equally saturating concentrations of VL9, B9 or Mtb44, to mimic the HLA-E cell-surface levels and CD94–NKG2A interaction capacity shown in FIGs. 17A and 17B. These cells were then incubated with the CD94–NKG2A Jurkat reporter cells for three- hours and measured Lucia® luciferase release to the media. As can be seen in FIG. 17F, at saturating concentrations, and in agreement with FIG. 17D, both Mtb44 and B9 reduced Lucia® luciferase secretion to approximately 20% of the VL9 control, indicative of markedly reduced cell-cell interactions through the HLA-E/B2M:CD94–NKG2A axis. Materials and Methods [0303] FACS analysis: SCaBER cells, a Squamous Cell Carcinoma cell line [ATCC HTB-3], were made as HLA-E KO (Cerulean BFP) and a NTC (Venus YFP) cell lines [information about this]. Cells were routinely cultured in DMEM [Corning 10-017-CV] + 10% FBS [Gibco] that has been heat-inactivated for 30 min at 56C in Corning® CellBIND® 75cm2 flasks [Corning 3293] at 37°C (5% CO2, 20% O2, humidity controlled). Media was sterile filtered before use. To prepare for the assay, cells were plated at 0.1e6 cells per well in a Falcon® 12-well plate [Corning 353043] with IFN-γ [Stemcell Technologies # 78020] stimulation (20 ng/mL) overnight. To pulse the cells, VL9 was diluted to 1mM in cell culture-grade water [Corning 25- 055-CM] and then added to serum-free DMEM at a final concentration of 20μM. Cells were washed twice with 1X PBS and then VL9-media was added to cells. The plate was incubated at 37C for 1 hr. Candidate peptides and their controls were diluted to 1mM in tissue-grade water and then diluted to 20μM in serum-free DMEM. VL9 was spiked into the media containing candidate peptides and controls at a final concentration of 2.5μM to allow for competition and high surface HLA-E levels. After VL9 pulsing, cells were washed twice with 1X PBS and the peptide-containing media added to the wells and the plate incubated at 37°C for 2hrs. After exchange, the cells were washed twice with 1X PBS and then rinsed with Accutase® solution [Sigma A6964-100ML]. The plate was returned to 37°C for 10min to allow for proper detachment from the plate. The detached cells were rinsed from the plate with BD Pharmingen™ Stain Buffer (BSA) [BD Biosciences 554657] FCS staining buffer. [0304] Cells were split into 2 wells of a 8-well PCR Strip tube (Axygen) and spun down at 1300 rpm for 5 min at room temperature. Blocking buffer was prepared by adding BD Pharmingen™ Human BD Fc Block™ (BD Biosciences 564219) to Pharmingen Stain Buffer at a 1:20 v/v dilution. The supernatant was aspirated and 100uL of Blocking buffer added to the wells and the 103 IPTS/128777599.4 Attorney Docket No.: CLS-039WO cells gently resuspended. The tubes were incubated on ice for 30 min. Staining buffers were made while the cells block by diluting APC anti-human HLA-E Antibody (Clone 3D12; Biolegend 342606) to 1:20 in Pharmingen Stain Buffer. Biotinylated Human NKG2A&CD94 Protein, Fc, Avitag™ (Acro Biosystems NC4-H82F5) was diluted to 200nM in Pharmingen Stain Buffer and then Streptavidin, Alexa Fluor™ 647 Conjugate (Invitrogen S21374) added at 50nM to allow for tetramerization of the complex before staining. A mouse isotype control, APC Mouse IgG1, κ Isotype Ctrl (FC) Antibody (Clone MOPC-21; BioLegend 400122), and Streptavidin AF647-only control were also included at respective concentrations. After blocking, cells were spun down at 1300 rpm for 5 min at 4C and the supernatant aspirated. Stains were added individually to the tubes and the cells gently resuspended. Cells were incubated for 1 hr at 4°C for 1 hr. After staining, cells were spun down and washed twice with Pharmingen Stain Buffer at the same speed and temperature as above. Cells were fixed with 4% Paraformaldehyde (Invitrogen 047340.9M) in 1X PBS for 30 min at 4C for 30 min. Cells were spun down and then resuspended in 200uL Pharmingen Stain Buffer in a 96-well plate for FACS analysis on a BD LSRFortessa™ X-20 Cell Analyzer. APC voltages were set to isotype and Streptavidin AF647- only controls. Data was analyzed using FlowJo™ software. [0305] Jurkat Reporter Assay: SCaBER NTC (Venus YFP) were plated in a Falcon® 96-well plate (Corning 353072) with IFN-γ (20 ng/mL) in DMEM + 10% FBS overnight at 37°C. IFN-y was persistent throughout at a concentration of 20 ng/mL. VL9 peptide was diluted to 1mM in cell culture grade water and then diluted in serum-free DMEM to a final concentration of 20μM. The plate was washed twice with 1X PBS and the VL9 media was added and the plate incubated at 37°C for 1 hr. Inhibitory peptides were diluted to 1mM in cell culture grade water and then added to serum-free DMEM (containing 10μM VL9) to a final concentration of 50μM. Peptides were serial diluted 1:2. VL9 was diluted to 20μM in serum-free DMEM and serial diluted down to generate a standard curve for Jurkat Reporter interaction. Plates were incubated at 37°C for 2 hr. Jurkat Reporter cells (Jurkat E6.1 cells harboring a CAR expression cassette consisting of CD3z-CD28-NKG2A-(ITIM-mut)-P2A-CD94 linked to an NFAT-Lucia secreted luciferase) were routinely cultured in RPMI1640 (Cytiva Life Sciences SH30096.01) + 10% FBS (heat- inactivated). Jurkat Reporter cells were spun down at 1300 rpm, washed once with 1X PBS, and then resuspended in serum-free DMEM to a concentration of 15e6/mL. After the 2 hr exchange, 20μL of the Jurkat Reporter cells were added to each well to a final concentration of 300K/well. The plates were spun down at 500 rpm for 1 min to settle the cells and the plate was incubated 104 IPTS/128777599.4 Attorney Docket No.: CLS-039WO for 3 hrs at 37°C. Conditioned media was harvested from the cells and spun down at 1300 rpm for 5 min to clear any cell debris. The cleared conditioned media (50μL) was added to a white- walled low-bind 96-well plate. Quanti-Luc Gold (InvivoGen rep-qlcg5) was dissolved in 25mL of sterile water to make the coelenterazine-based luminescence detection reagent for Lucia. To each well, 50μL was added and the luminescence measured at Em480nm on a Clariostar plate reader. Data was analyzed in Prism. EXAMPLE 13 – Synthesis of Lipidated Peptide [0001] This example describes the synthesis of an exemplary lipidated peptide to HLA-E. Because the crystal structure of an exemplary peptide, B9, bound to HLA-E suggested that the fluorophenylalanine in position 5 was solvent-exposed and not involved in any major binding interactions, the position was deemed suited for the introduction of a lipid side chain. Accordingly, a B9 analog featuring an Alloc-protected Lys in position 5 was synthesized using standard Fmoc SPPS protocols. Following palladium-mediated Alloc deprotection, standard Fmoc-SPPs was utilized to couple two PEG2 linkers, γ-Glu, and C18 octadecanedioic acid. Subsequent cleavage and purification afforded the lipidated B9 peptide candidate. See FIG. 9 for synthesis schematic. That lipidated B9 peptide candidate is also referred to as “B95K lipid”. A control peptide with a scrambled sequence was prepared analogously. [0002] Lipidation was also performed at a Lys-26 position in an HLA-E targeted peptide. Systematic optimization of the lipid chain during semaglutide development revealed consistently positive effects with a C18 octadecanedioic acid group attached to Lys-26, along with an extended spacer unit composed of γ-Glu linked to two 8-amino-3,6-dioxaoctanoic acid groups EXAMPLE 14 – Characterization of Lipidated Peptides [0003] This example describes the characterization of an exemplary lipidated peptide to HLA-E, lipidated B9. A comparison of the candidate lipidated peptide and scramble control showed the results in TABLE 20. See also FIGs. 10A and 10B for the structures and indicated chromatograms. Table 20.
Figure imgf000107_0001
Stability Assays 105 IPTS/128777599.4 Attorney Docket No.: CLS-039WO [0004] Stability assays were conducted and showed improved serum stability for the lipidated B9 peptide, as shown in FIG. 11. B9 and lipidated B9 were incubated in 25% human serum or 10% FBS for 24 hours and monitored by LC-MS at regular intervals in the presence of a reference peptide (heavy B9). Heavy B9 contains a heavy (isotope) version of Glutamine (Gln, 13C5/15N2, +7 ) purchased from Cambridge Isotope Laboratories, Catalog No. CNLM-7252-H-PK. While the area under the curve (AUC) in the LC-MS decreases 100- fold within 30 min for B9 in HS and FBS, the lipidated version exhibits improved serum stability with a 10-fold improvement even after 24 hours. Protein-Based Assays [0005] The ability of the B9 lipidated peptide to exchange with HLA-E/B2M refolded with a FAM-labled VL9 peptide was compared to the parental B9 peptide. The results, as illustrated in FIG. 12, showed lipidated B9 peptide shares similar exchange rates to the B9 peptide in 1X PBS and 1X PBS + 1% BSA buffers. When 10% FBS was added, the B9 lipidated peptide continued exchanging over the course of the assay, whereas the B9 peptide becomes exhausted early on in the assay. Without being bound by mechanism of action, it is possible that the B9 peptide is becoming sequestered by components in the FBS (not by BSA alone) and that the lipidation of the peptide helps keep it available for exchange. [0006] Thereafter, to determine the ability of the peptide to be inhibitory towards CD94-NKG2A binding to HLA-E/peptide/B2M complex, peptides were incubated with HLA- E/VL9/B2M*Biotin overnight in 1X PBS, 1X PBS + 1% BSA, or 1X PBS + 10% FBS. Then HLA-E/peptide/B2M complex was adsorbed on a SAX BLI sensor in solution and measured association/dissociation of a CD94-NKG2A heterodimeric complex. The results are shown in FIG. 13. The B9 lipidated peptide, again, was able to be inhibitory towards the CD94-NKG2A co-receptor in the presence of FBS, where the parental B9 peptide loses efficacy of inhibition. INCORPORATION BY REFERENCE [0007] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS [0008] An invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on any invention disclosed herein. Scope of an invention is thus indicated by the appended claims rather than by the foregoing description, and 106 IPTS/128777599.4 Attorney Docket No.: CLS-039WO all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. SEQUENCE LISTING SUMMARY
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118 IPTS/128777599.4

Claims

Attorney Docket No.: CLS-039WO CLAIMS What is claimed is 1. A synthetic peptide comprising an amino acid sequence X1-Met-X3-X4-X5-Ala-X7-X8- Leu (SEQ ID NO: 3), wherein each of X1, X3, X4, and X7 are independently selected from 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg; X5 is Ala, Cha, Tha, or Mff; and X8 is Arg, Msn, or hAr; wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 2. The synthetic peptide of claim 1, wherein the amino acid sequence is (a) NH2-4Af-Met-Dff-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 5); (b) NH2-hAr-Met-Msn-Dff-Cha-Ala-Arg-Msn-Leu-OH (SEQ ID NO: 6); (c) NH2-Ala-Met-hAr-Dff-Tha-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 7); (d) NH2-4Af-Met-Ala-Ser-Ala-Ala-Cha-Arg-Leu-OH (SEQ ID NO: 8); or (e) NH2-hAr-Met-Har-Gln-Mff-Ala-Cha-hAr-Leu-OH (SEQ ID NO: 9). 3. The synthetic peptide of claim 1 or claim 2, wherein one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. 4. The synthetic peptide of claim 3, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. 5. The synthetic peptide of claim 4, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys. 6. The synthetic peptide of claim 4, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 and is substituted with a Cys. 7. The synthetic peptide of any one of claims 3 to 6, wherein the one or more Cys, Lys, Tyr, His, Ser, or Thr is arylated. 8. The synthetic peptide of any one of claims 3 to 6, wherein the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead. 119 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 9. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000122_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, - C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, - OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, - N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen,- OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 10. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000122_0002
, wherein R2 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) 120 IPTS/128777599.4 Attorney Docket No.: CLS-039WO N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 11. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000123_0001
, wherein R3 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 12. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000123_0002
121 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein R10 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 13. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000124_0001
wherein X is a halogen. 14. The synthetic peptide of claim 8, wherein the warhead is
Figure imgf000124_0002
. 15. The synthetic peptide of claim 8, wherein the warhead is selected from the group consisting of 122 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000125_0001
16. The synthetic peptide of claim 8, wherein the warhead is selected from the group of a sulfonyl fluoride, a phenyl carbamate, and a squaramate. 17. The synthetic peptide any one of claims 8 to 16, wherein the warhead is conjugated to the Cys via the sulfur atom of the Cys. 18. A synthetic peptide comprising an amino acid sequence hAr-X2-hAr-Gln-Mff-A-Cha- hAr-X9 (SEQ ID NO: 20) wherein X2 is Nle or Mox; and X9 is Leu, Aoa, or Cha; wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 19. The synthetic peptide of claim 18, wherein the amino acid sequence is 123 IPTS/128777599.4 Attorney Docket No.: CLS-039WO (a) NH2-hAr-Nle-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (SEQ ID NO: 21); (b) NH2-hAr-Mox-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (SEQ ID NO: 22); (c) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Nle-OH (SEQ ID NO: 23); (d) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Aoa-OH (SEQ ID NO: 24); or (e) NH2-hAr-Met-hAr-Gln-Mff-Ala-Cha-hAr-Cha-OH (SEQ ID NO: 25). 20. The synthetic peptide of claim 18, wherein the amino acid sequence is NH2-hAr-Nle-hAr-Gln-Mff-Ala-Cha-hAr-Leu-OH (SEQ ID NO: 21). 21. The synthetic peptide of claim 19 or 20, wherein the Lys-Ry substitution is at position 5. 22. The synthetic peptide of claim 18 or claim 19, wherein one or more other amino acids is substituted with a Cys, Lys, Tyr, His, Ser, or Thr. 23. The synthetic peptide of any one of claims 18, 19, and 22, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. 24. The synthetic peptide of claim 23, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cys. 25. A synthetic peptide comprising the formula
Figure imgf000126_0001
wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 26. A synthetic peptide comprising the formula 124 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000127_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, - C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, - OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, - N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, - OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl; and wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 125 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 27. A synthetic peptide comprising an amino acid sequence VMAPRT(L/V)(V/L/I/F)L, wherein one or more amino acids is substituted with a Lys-Ry; wherein the Ry comprises C18 octadecanedioic acid and a linker; and wherein one or more other amino acids are substituted with a Cys, Lys, Tyr, His, Ser, or Thr. 28. The synthetic peptide of claim 27, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 3 or position 8. 29. The synthetic peptide of claim 28, wherein the Cys, Lys, Tyr, His, Ser, or Thr substitution is at position 8 and is substituted with a Cysteine. 30. The synthetic peptide of any one of claims 27 to 29, wherein the peptide has the amino acid sequence VMAPRTLFL. 31. The synthetic peptide of any one of claims 27 to 30 wherein the one or more Cys, Lys, Tyr, His, Ser, or Thr is conjugated to a warhead. 32. The synthetic peptide of claim 31, wherein the warhead is
Figure imgf000128_0001
wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, - C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, - OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, - N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, - 126 IPTS/128777599.4 Attorney Docket No.: CLS-039WO OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 33. The synthetic peptide of claim 31, wherein the warhead is
Figure imgf000129_0001
, wherein R2 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 34. The synthetic peptide of claim 31, wherein the warhead is
Figure imgf000129_0002
, wherein R3 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl 127 IPTS/128777599.4 Attorney Docket No.: CLS-039WO OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 35. The synthetic peptide of claim 31, wherein the warhead is
Figure imgf000130_0001
wherein R10 is hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, -C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, - OC(=O)RC3, -OC(=O)ORC3, -OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, -OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, - N(RC3)C(=NRC3)RC3, -N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, -N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 36. The synthetic peptide of claim 31, wherein the warhead is 128 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000131_0001
wherein X is a halogen. 37. The synthetic peptide of claim 31, wherein the warhead is
Figure imgf000131_0002
. 38. The synthetic peptide of claim 31, wherein the warhead is selected from the group consisting of
Figure imgf000131_0003
129 IPTS/128777599.4 Attorney Docket No.: CLS-039WO ,
Figure imgf000132_0001
39. The synthetic peptide of any one of claims 31 to 38, wherein the warhead is conjugated to the Cys via the sulfur atom of the Cys. 40. A synthetic peptide comprising the formula
Figure imgf000132_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 41. A synthetic peptide comprising the formula 130 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000133_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 42. A synthetic peptide comprising the formula
Figure imgf000133_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 43. A synthetic peptide comprising the formula
Figure imgf000133_0003
, 131 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 44. A synthetic peptide comprising the formula
Figure imgf000134_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 45. A synthetic peptide comprising the formula
Figure imgf000134_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 46. A synthetic peptide comprising the formula
Figure imgf000134_0003
, 132 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker 47. A synthetic peptide comprising the formula
Figure imgf000135_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 48. A synthetic peptide comprising the formula
Figure imgf000135_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 49. A synthetic peptide comprising the formula 133 IPTS/128777599.4 Attorney Docket No.: CLS-039WO
Figure imgf000136_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 50. A synthetic peptide comprising the formula O F
Figure imgf000136_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 134 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 51. A synthetic peptide comprising the formula
Figure imgf000137_0001
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 52. A synthetic peptide comprising the formula S
Figure imgf000137_0002
, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 53. A synthetic peptide comprising the formula
Figure imgf000137_0003
S O O , 135 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 54. A synthetic peptide comprising the formula O F
Figure imgf000138_0001
wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 55. A synthetic peptide comprising the formula
Figure imgf000138_0002
S O O , wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker. 56. The synthetic peptide of any one of claims 48 to 55, wherein R is
Figure imgf000138_0003
136 IPTS/128777599.4 Attorney Docket No.: CLS-039WO wherein R1, R2, and R3 are each independently hydrogen, halogen, -CN, -NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -ORC3, -C1-C6 alkyl N(RC3)2, -N(RC3)2, -SRC3, -C(=O)RC3, - C(=O)ORC3, -C(=O)SRC3, -C(=O)N(RC3)2, -OC(=O)RC3, -OC(=O)ORC3, - OC(=O)N(RC3)2, -OC(=O)SRC3, -OS(=O)2RC3, -C1-C6 alkyl OS(=O)2RC3, - OS(=O)2ORC3, -OS(=O)2N(RC3)2, -N(RC3)C(=O)RC3, -N(RC3)C(=NRC3)RC3, - N(RC3)C(=O)ORC3, -N(RC3)C(=O)N(RC3)2, -N(RC3)C(=NRC3) N(RC3)2, - N(RC3)S(=O)2RC3, -N(RC3)S(=O)2ORC3, -N(RC3)S(=O)2N(RC3)2, -SC(=O)RC3, - SC(=O)ORC3, -SC(=O)SRC3, -SC(=O)N(RC3)2, -S(=O)2RC3, -S(=O)2ORC3, or - S(=O)2N(RC3)2, wherein each instance of RC3 is independently selected from hydrogen, - OPh, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. 57. The synthetic peptide of any one of claims 1 to 56, wherein the synthetic peptide is an HLA-E-NKG2A complex specific inhibitor. 58. A synthetic peptide comprising the amino acid sequence of VMAPRTLFL, wherein one or more amino acids is substituted with a Lys-Ry; and wherein the Ry comprises C18 octadecanedioic acid and a linker; and further comprising one or more other amino acid substitution, 59. The synthetic peptide of claim 58, wherein the one or more other amino acid substitution comprises: a) a substitution of the V residue at a position 1; b) a substitution of the M residue at a position 2; c) a substitution of the A residue at a position 3; d) a substitution of the P residue at a position 4; e) a substitution of the R residue at a position 5; f) a substitution of the T residue at a position 6; 137 IPTS/128777599.4 Attorney Docket No.: CLS-039WO g) a substitution of the L residue at a position 7; h) a substitution of the F residue at a position 8; i) a substitution of the L residue at a position 9; or a combination of any of the foregoing substitutions. 60. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 1 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. 61. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 3 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. 62. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 4 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. 63. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 5 and the amino acid is substituted for an Ala, Cha, Tha, or Mff. 64. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 7 and the amino acid is substituted for a 4Af, hAr, Ala, Dff, Ser, Msn, Gln, Cha, or Arg. 65. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 8 and the amino acid is substituted for a Arg, Msn, or hAr. 66. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 3 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. 67. The synthetic peptide of any one of claims 58 or 59, wherein the substitution is at position 8 and the amino acid is substituted for a Cys, Lys, Tyr, His, Ser, or Thr. 68. The synthetic peptide of any one of claims 1-67, wherein the linker comprises a PEG linker. 138 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 69. The synthetic peptide of any one of claims 1-67, wherein the linker comprises a PEG2 linker. 70. The synthetic peptide of claim 69, wherein the linker comprises two PEG2 linkers. 71. The synthetic peptide of any one of claim 69 or 70, where the PEG2 linker is 8-amino- 3,6-dioxaoctanoic acid. 72. The synthetic peptide of any one of claims 1-71, wherein the linker comprises a γ-Glu. 73. A synthetic peptide comprising the formula
Figure imgf000141_0001
74. A synthetic peptide of any one of claims 1-73 comprising one or more additional modifications selected from: a) acetylated, formylated, propanoylated, hexanoylated, or myristoylated N-terminus; b) amidated C-terminus; c) substitution of one or more L-amino acid with a D-amino acid; d) substitution of one or more amino acid with a methyl-amino acid; and e) substitution of an α-amino acid with a β-amino acids. 75. The synthetic peptide of any one of claims 1-74, wherein the synthetic peptide increases NK-mediated cell killing, optionally as measured by a luciferase-based assay. 139 IPTS/128777599.4 Attorney Docket No.: CLS-039WO 76. The synthetic peptide of any one of claims 1-74, wherein the synthetic peptide reduces interaction of HLA-E with CD94/NKG2A, optionally relative to a comparator (e.g., VL9, Mtb44, etc.). 77. The synthetic peptide of any one of claims 1-74, wherein the synthetic peptide inhibits CD94/NKG2A binding to HLA-E, optionally relative to a comparator (e.g., VL9, Mtb44, etc.). 78. A synthetic peptide/HLA-E complex, wherein the peptide is selected from any one of the synthetic peptides of claims 1 to 74. 79. The complex of claim 78, wherein the synthetic peptide and the HLA-E in the complex are covalently linked. 80. The complex of any one of claims 78 or 79, wherein the HLA-E is human HLA-E. 81. The complex of any one of claims 78 to 80, wherein the synthetic peptide is covalently linked to amino acid residue Tyr-7, Lys-146, Tyr-159, or Tyr-171 of human HLA-E. 82. The complex of any one of claims 78 to 81, wherein the synthetic peptide is covalently linked to an amino acid residue selected from the group of Tyr-7, His-9, Ser-24, Tyr-59, Arg-62, Glu-63, Ser-66, Thr-70, Gln-72, Asn-77, Thr-80, Tyr-84, Trp-97, His-99, Glu- 114, Tyr-123, Trp-133, Ser-143, Lys-146, Ser-147, Glu-152, His-155, Gln-156, Tyr-159, Thr-163, Cys-164, Trp-167, and Tyr-171 of human HLA-E. 83. The HLA-E peptide complex of any one of claims 78 to 82, wherein the complex is inhibited in binding of CD94/NKG2A or prevents activation of CD94/NKG2A. 84. A pharmaceutical composition, comprising the synthetic peptide of any one of claims 1- 74 and a pharmaceutically acceptable salt or carrier. 140 IPTS/128777599.4
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