De novo designed protein receptors for peptide-MHC complexes

Abstract

Polypeptides having an amino acid sequence at least 50% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59, wherein the polypeptide binds to a target peptide-MHC complex are provided, fusion proteins thereof, and methods for their use.

Description

[0001] De Novo Designed Protein Receptors for Peptide-MHC Complexes

[0002] Sequence Listing Statement

[0003] The instant application contains an electronic Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on March 24, 2026, is named “25-0495-WO_ST26.xml” and is 103,612 bytes in size.

[0004] Background

[0005] MHC-I molecules are cell-surface proteins that present peptides derived from intracellular proteins. The recognition of peptides displayed on MHC-I by the T-cell receptor enables the immune system to detect foreign proteins within cells and destroy tumor and viral infected cells. From the therapeutic perspective, targeting pMHCs with engineered cells or proteins-for example, Bispecific T cell Engagers (BiTEs) is attractive as it provides a unique opportunity to distinguish cells based on their intracellular proteins. T-cell receptors can be used for such targeting, but TCRs with the necessary specificity have not been identified for many targets of therapeutic interest, engineering TCRs with new specificities has been very challenging, and for protein based therapeutic strategies, TCR extracellular domains are quite difficult to produce in soluble form. The diversity of MHC alleles and antigenic peptides across patient populations necessitates the development of hundreds to thousands of effective and specific binders to achieve broad patient coverage. Current methods rely on empirical TCR screening from patient samples or single-chain variable fragment (scFv) libraries, are costly, labor-intensive, and time-consuming, making them economically infeasible for covering all pMHC targets, especially for smaller patient subpopulations. Methods for rapidly generating small stable proteins that recognize specific pMHCs of interest could have considerable therapeutic utility as the recognition domains in both chimeric antigen receptors (CARs) for cell-based therapies, and in protein-based therapies such as BiTEs.

[0006] Summary

[0007] In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59, wherein the polypeptide binds to a target peptide-MHC complex as noted in Table 1.

[0008] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In another embodiment, at least 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, or all identified interface residues are identical (not substituted), relative to the reference sequence. In a further embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In a further embodiment, all interface residues and all specific residues are identical (not substituted), relative to the reference sequence. In another embodiment, substitutions relative to the reference sequence are conservative amino acid substitutions.

[0009] In another embodiment, the disclosure provides fusion proteins, comprising:

[0010] (a) the polypeptide of any embodiment or combination of embodiments herein; and

[0011] (b) one or more functional domains at the N-terminus and / or at the C-terminus of the polypeptide.

[0012] In one embodiment, the fusion protein comprises a chimeric antigen receptor (CAR). In one embodiment, the fusion protein comprises the formula X1-X2-X3-X4, wherein XI comprises the polypeptide of embodiment of combination of embodiments herein; X2 comprises a hinge domain;

[0013] X3 comprises a transmembrane domain;

[0014] X4 comprises an intracellular domain;

[0015] optionally including an amino acid linker between 1, 2, or all 3 of X1-X2; X2-X3; and X3-X4.

[0016] In one embodiment, one or more of the following is true:

[0017] (i) the hinge domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:43;

[0018] (ii) the transmembrane domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44; and / or(iii) the intracellular domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, linked in N- to C-terminal order.

[0019] In a further embodiment, the fusion protein comprises the formula Z1-Z2-Z3, wherein (a) Z1 comprises the amino acid sequence at least 50%, 75%, 90%, or 100% identical to the amino acid sequence of SEQ ID NO: 60;

[0020] (b) Z2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the polypeptide of any embodiment or combination of embodiments herein; and

[0021] (c) Z3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61.

[0022] In another embodiment, the fusion protein comprises a bi-specific T-cell engager of the general formula X1-X2-X3, wherein XI comprises the polypeptide of any embodiment or combination of embodiments herein, X2 comprises an Fc CH2 domain, and X3 comprises an Fc CH3 domain, wherein XI, X2 and X3 can be in any order in the fusion protein.

[0023] In one embodiment, the domain order is X1-X2-X3. In another embodiment

[0024] (a) X2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and

[0025] (b) X3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55

[0026] In a further embodiment, the fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52.

[0027] The disclosure also provides dimers, comprising

[0028] (a) the bi-specific T-cell engager fusion protein of any embodiment or combination of embodiments herein; and

[0029] (b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52 or 53.In another embodiment, the disclosure provides kits comprising:

[0030] (a) the bi-specific T-cell engager fusion protein of any embodiment or combination of embodiments herein; and

[0031] (b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53.

[0032] In another aspect the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure. In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence. In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic.

[0033] The disclosure also provides pharmaceutical compositions, comprising:

[0034] (a) the polypeptide, the fusion protein, the nucleic acid, the expression vector, the host cell, the kit, and / or the dimer of any embodiment or combination of embodiments herein; and

[0035] (b) a pharmaceutically acceptable carrier.

[0036] In another aspect, the disclosure provides methods for treating or limiting a disorder associated with a target pMHC in Table 1, comprising administering to a subject in need thereof an amount effective to treat or limit development of the disorder of the polypeptide, fusion protein, nucleic acid, expression vector, cell, dimer, or pharmaceutical composition of any embodiment or combination of embodiments herein.

[0037] Description of the Figures

[0038] Figure 1. Diffusion of pMHC binders. (A) pMHC structure and design challenge. The goal is to distinguish a target peptide (in this example, MAGE) from a closely related off-target (Titin). The positions that differ between the two peptides are circled in the pMHC structure model at the bottom. (B) Representative diffusion trajectories and design models for three different pMHC targets. Column 1 target pMHC, column 2 initial gaussian noise initialization. Columns 3 and 4, intermediate steps in diffusion denoising trajectories starting from completely random residue distributions. Right column, fully denoised design model backbones. (C) Partial diffusion of design scaffolds with desirable properties (interacting overthe full length of the peptide but making few interactions with MHC) to more efficiently generate binders to related targets.

[0039] Figure 2. Generation of recyclable scaffold library to peptide-MHC structures.

[0040] (A) Examples of diverse diffusion scaffolds on pMHC target. (B) Designs are filtered based on peptide contact area. Designs with extensive peptide contacts are selected, while those with limited peptide contacts are filtered out. (C) Target peptide residue mutation effect evaluation process by ProteinMPNN™ using predicted binder-pMHC structures. (D) Plot of pAE interaction values using AF-MHC to fold each design against MHC loaded with target peptides or off-target peptides. Filter (on low interaction pAE interaction for on-target, high pAE interaction for off-target.

[0041] Figure 3. Design models and binding specificity. (A) Design models. Left, overall structure; right, zoom in on peptide binding region. HLA allele and peptide sequence are specified above the zoom-in view. (B) Flow cytometry of cells displaying the design incubated with on-target pMHC tetramer (x-axis) and 1 or 2 off-target tetramers (y-axis) at lOnM concentration. Staining in the lower right quadrant indicates specific on-target binding. Rows 1 and 2, individual designs displayed on yeast; rows 3-6, CARs incorporating designs on Jurkat cells. From top to bottom, sequences are (Y-axis) GLAWLSLFV (SEQ ID NO: 73), GLIFSSYFV (SEQ ID NO: 74), by (X-axis) GLMWLSYFV (SEQ ID NO: 72); (Y-axis) IVWNGPVG (SEQ ID NO: 97), TLWRGPVVV (SEQ ID NO: 78) by (X-axis) LLWNGPIAV (SEQ ID NO: 76); (Y-axis) KLTTVCPTV (SEQ ID NO: 98), KLIPLCHQL (SEQ ID NO: 67) by (X-axis) KLTPLCVTL (SEQ ID NO: 64); (Y-axis) HLYPNTPYA (SEQ ID NO: 70), RLFPNLPEL (SEQ ID NO: 69) by (X-axis) RMFPNAPY (SEQ ID NO: 99); (Y-axis) TLMSMVANL (SEQ ID NO: 86), TLFSALTGL (SEQ ID NO: 87) by (X-axis) TLMSAMTNL (SEQ ID NO: 85); (Y-axis) TTAPSLSGK (SEQ ID NO: 94) by (X-axis) TTAPFLSGK (SEQ ID NO: 93). (C) Partial diffusion of design at top to targets below (left panels) and corresponding Jurkat straining (right). From top to bottom, sequences are (Y-axis) ESDPIVAQY (SEQ ID NO: 63) by (X-axis) EVDPIGHLY (SEQ ID NO: 62); (Y-axis) FQDPVPLTV (SEQ ID NO: 83), YLQPWPVDV (SEQ ID NO: 84) by (X-axis) YLEPGPVTA (SEQ ID NO: 82); (Y-axis) SLAGLGLWLL (SEQ ID NO: 81) by (X-axis) ELAGIGILTV (SEQ ID NO: 79); (Y-axis) ILYVDPLPMI (SEQ ID NO: 89) by (X-axis) ALYVDSLFFL (SEQ ID NO: 88)

[0042] Figure 4. Yeast surface display library screening and biochemical characterization of individual hits. (A) WT1 yeast surface display library screening; initial sort for surface expression followed by 3 binding sorts with target pMHC tetramer (y-axis,with decreasing concentrations for later sort) and unrelated or related off-target peptides pMHC tetramers (x-axis). Population in on-target gates was taken for subsequent sorting rounds. (B and C) SEC trace, SPR binding kinetics (binding affinity indicated in plot), and design model overlaid with prediction for HIV-10 design against A*02:01-KLTPLCVTL (B) and MAGE-513 design against A*01:01-EVDPIGHLY (C) purified from E. coli expression.

[0043] Figure 5. Selective activation of T-cells expressing designed CARs targeting MAGE-A3. Activation of Jurkat cells expressing the MAGE-513 CAR by 293T cells expressing HLA-A*01:01 pulsed with 5uM of different peptides measured through CD69 expression level. (A) Histograms of CD69 expression levels following treatment with 5uM MAGE, the closely related Titin peptide or DMSO; the MAGE peptide leads to considerable activation while the Titin peptide is similar to the DMSO control. Horizontal black bars represent CD69 positive population; the fraction of cells within this range is indicated at top left of each panel. (B) Design models of the MAGE-513 / MAGE peptide / HLA-A*01:01 complex (lower panel) and zoom-in view of the key residues mediating the interaction (upper panels). (C) Histograms of CD69 expression levels following pulsing with MAGE-A3 single alanine mutants (D3 A indicates mutation of the Asp at position 3 in the peptide to alanine) or DMSO. (D) Histograms of CD69 expression levels following pulsing MAGE-513 variant CARs with MAGE-A3 peptide or DMSO. (E and F) CD69 levels of Jurkat cells expressing MAGE-513 CAR upon pulsing with top ranked peptides from yeast binding screen (E) or from sequence similarity search (F). Sequences on the X-axis of (E) are EVDPIGHLY (SEQ ID NO: 62), MVDPIIPLY (SEQ ID NO: 100), TSDFSMSFY (SEQ ID NO: 101), YLEAIKPLY (SEQ ID NO: 102), ETDFRWWTY (SEQ ID NO: 103), RVDFYVGMY (SEQ ID NO: 104), RIDPVRSLY (SEQ ID NO: 105), RVDFYVGMY (SEQ ID NO: 106), VSDFRWWTY (SEQ ID NO: 107), ITDFRWWTY (SEQ ID NO: 108), ESDFNWWSY (SEQ ID NO: 109), and ESDPIVAQY (SEQ ID NO: 63). Sequences on the X-axis of (F) are EVDPIGHLY (SEQ ID NO: 62), EVDPIGHVY (SEQ ID NO: 95), VTDFISHLF (SEQ ID NO: 96), EMDPVTQLY (SEQ ID NO: 110), EFDPICALF (SEQ ID NO: 111), EFEPVFSLF (SEQ ID NO: 112), ESDPIVAQY (SEQ ID NO: 63), ELDPIQKLF (SEQ ID NO: 113), EAEPRQHLY (SEQ ID NO: 114), and SSDPIWDLY (SEQ ID NO: 115). Identity to the MAGE peptide is indicated.

[0044] Figure 6. Binding and activation of different MAGE-A3 binder based CAR. (A) Flow cytometry of jurkat cells displaying the design expressing 3 MAGE- A3 binders (MAGE-282, MAGE-513, hit-4) or CD19 scFV (negative control) based CAR incubated with on-target MAGE- A3 pMHC tetramer (x axis) and off-target Titin tetramers (y axis) at lOnMconcentration. (B) Histograms of CD69 level of Jurkat cells expressing 3 MAGE-A3 binders (MAGE-282, MAGE-513, hit-4) or CD19 scFV (negative control) based CAR incubated with 293T or HLA-A*01:01 expressing 293T upon pulsing with 5uM indicated peptides (MAGE-A3, Titin) orDMSO.

[0045] Figure 7. Biochemical and cellular characterization of the PRAME A2 and HIV_10. (A) CD69 MFI of Jurkat cells expressing indicated CARs (in each column) incubated with 293T upon pulsing with 5uM respective peptide (top) orDMSO (bottom). (B) SPR traces of PRAME binder A2 on PRAME pMHC monomer. (C) zoom-in view of PRAME A2 L36 close distance with S6 of PRAME peptide form design model of PRAME A2 / PRAME peptide / HLA-A*02:01. (D) CD69 MFI of Jurkat cells expressing PRAME binder A2 (left) and A2 L36G (right) CARs incubated with 293T upon pulsing with 5uM PRMAE peptide, alanine mutant peptides, four peptides with similar sequences, or DMSO. (E) CD69 MFI of Jurkat cells expressing HIV binder 10 CAR incubated with 293T upon pulsing with 5uM HIV peptide, alanine mutant peptides, three peptides with similar sequences, or DMSO.

[0046] Figure 8. Specific activation of designed CARs by cognate pMHC complexes. Activation of Jurkat cells expressing the CARs by 293T cells with 5uM of different peptides measured through CD69 expression level by staining (indicated by histogram or mean fluorescence intensity (MFI)). (A) CD69 MFI of gpl00_T3 CAR with gplOO peptide, alanine mutant peptides or DMSO. (B) CD69 MFI of WT1 5 CAR with WT1 peptide, alanine mutant peptides, off-target peptides, or DMSO. (C) zoom-in view of design model of WT1 5 with pMHC target. (D and E) (upper) CD69 MFI of (D) Mart- 1 3 or (E) Mart-1 43 CAR with Mart-1 peptide, alanine mutant peptides, or DMSO. (lower) design models of binders with Mart-1 pMHC antigen. (F) Histograms of CD69 expression level of PRAME CARs with PRAME peptides or DMSO. (G) SPR traces of PRAME binder A9 on PRAME pMHC monomer. (H) CD69 MFI of Jurkat cells expressing PRAME A9 CAR with PRAME peptide, alanine mutant peptides, four peptides with similar sequences, or DMSO.

[0047] Detailed Description / Claims

[0048] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA),“Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

[0049] 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319-322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0050] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0052] Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides disclosed herein, 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and / or the C-terminus of the polypeptide while retaining activity.

[0053] All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

[0054] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0055] In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59, wherein the polypeptide binds to a target peptide-MHC complex as noted in Table 1.The polypeptides of the disclosure bind to specific peptide-MHC (pMHC) complexes as noted in Table 1 (See “target allele” and “target peptide”) and thus can be used for protein and cell based pMHC targeting to, for example treat disease associated with the target pMHC. Major histocompatibility complex (MHC)-I molecules are cell-surface proteins that present peptides derived from intracellular proteins. The recognition of peptides displayed on MHC-I by the T-cell receptor enables the immune system to detect foreign proteins within cells and destroy tumor and viral infected cells. The polypeptides of the disclosure may, for example, serve as the recognition domains in both chimeric antigen receptors (CARs) for cell based therapies, and in protein-based therapies such as bi-specific T-cell engagers.

[0056] By way of example, the polypeptide of SEQ ID NO: 1 targets a pMHC complex of MHC allele A*01 :01 complexed with target peptide EVDPIGHLY. Those of skill in the art will clearly understand from Table 1 the pMHC complexes that the other polypeptides of the disclosure selectively bind to.

[0057] Table 1. Binder sequences and targets

[0058] Bin Binder Target Target Target Binder AA Interface Specific der name allele peptide name sequence residues residues SEQ ID NO

[0059] 1 mage- A*01 : 01 EVDPIGH MAGE- DLVERLSERALRL V3 , S7 , Lil L14 , D40

[0060] 282 LY A3 LRRLPELGHEERQ , L14 ,A39 ,

[0061] (Melan VTEALARLYSTLA D40 , V43 , V

[0062] oma- DIAVEDERLGVDR 89 ,A92 ,V9

[0063] associ REELEQRLRELEK 3 , Q96

[0064] ated LEKAPLEERESAL

[0065] antige PELERELEELVER

[0066] n 3 ) AVKRQVKHFRS

[0067] 2 mage- A*01 : 01 EVDPIGH MAGE- LTVEEAIRLLLET 17 , LIO , LI L10 , D29 , L32 , 513 LY A3 QRRYPELGPEERQ 1 , Q14 , P22 R33 , S36 , D40 ,

[0068] (Melan VADGALRVASLLL , D29 , L32 , A89 ,A93 oma- DIIATRRLLGDDD R33 , S36, L

[0069] associ KELEKQLLKRIER 39 , D40 ,A4

[0070] ated LFTAPLPERRRLV 3 , R46, L47

[0071] antige EELEEPVLEEAAQ , L86 ,A89 ,

[0072] n 3 ) AARELVKREQQ A90 , A93 , V

[0073] 97

[0074] 4 sars-6 A*02 : 01 GLMWLSY SARS- SPEVERLEKELQK L22 , Q23 , P P24 , G26 , 127 ,

[0075] EV CoV SLTELAEALQPAG 24 ,A25 , G2 R28 , L29 , E30 , membra IRLEPEALLIDVR 6 , 127 , R28 P31 , L34 ,V43 , ne NRWVDVESVIEAL , L29 , E30 , V48 protei RHLVGVPEDLLER P31 , E32 , A

[0076] n TIRLIRELAEAS 33 , L34 , 13

[0077] 6 , D37 , R39

[0078] , N40 ,W42 ,

[0079] V43 , D44 , S

[0080] 47 ,V48 , E5

[0081] 0 , A51 , H54

[0082] , L55 , V56

[0083] 5 sars- A*02 : 01 GLMWLSY SARS- DERKKQLQKELEQ E23 , K25 , G T28 , D30 , L34 , 11 EV CoV ARKEAQRAAEEKG 26 , F27 , T2 152fL56

[0084]

[0085] membra FTVDLDGLLLEAR 8 , V29, D30ne YRGIPDERLREEI , D32 , G33 ,

[0086] protei REVLRHHPEVAEL L34 , L36 , E

[0087] n VIRAVELTRELE 37 , R39 , Y4

[0088] 0 , R41 , R47

[0089] , 152 , V55 ,

[0090] L56 , R57 , H

[0091] 58 , H59 , V6

[0092] 6

[0093] hiv-5 A* 02 : 01 KLTPLCV HIV GVFDTLEEAFRLT V2 , T5 , E8 , A9 , T13 , A16 , L TL envelo VEALRNDRSVSPE A9 , L12 , T1 17 , 131 , S35 , V pe EARRILETSLDVF 3 , E15 , A16 38 , T42 protei LATAEEHGEDVEK , L17 , N19 ,

[0094] n LRKRLEELWN 131 , T34 , S

[0095] 35 , D37 , V3

[0096] 8 , A41 , T42

[0097] hiv- 10 A* 02 : 01 KLTPLCV HIV SEVERLVEIARTS R5 , I 9 , T12 I 9 , S13 , N16 , T TL envelo ARNLLLTAEELVE , S 13 , R15 , 20 , A42 , S46 , A pe RGHPNAVELLEHL N16 , L19 , T 49 , 150 , E53 protei REALELSTEAIVE 20 , L35 , H3

[0098] n EARRLGAPQEALD 8 , L39 , E41

[0099] EIRRVAEEALKRL , A42 , L45 , EELARRARES S 46 , E48 , A

[0100] 49 , 150 , E5

[0101] 3

[0102] wtl- 4 A* 02 : 01 RMFPNAP Wilms TEERRTVLVIPEF T 6 , V7 , L8 , T 6 , L8 , P11 , T2

[0103] YL tumor EDPHEKELAEEAL V9 , P11 , E1 9 , A32 , L33 antige KLTEEALLLDARY 8 , L21 , E24

[0104] n 1 DGHTGVTLTREEE , A25 , L28 ,

[0105] (WT1 ) EVEIELPDGTVEK T29 , E31 , A IRIVTITLTT 32 , L33 , L3

[0106] 5 , R38 , Y39

[0107] , 156 , L58

[0108] wtl-5 A* 02 : 01 RMFPNAP Wilms LTPEERQRREAIV R8 , A11 , I l T19 , L23 , T47

[0109] YL tumor ESVRLTLEALELD 2 , E14 , S15

[0110] antige IEYAEREGFDLRA , L18 , T19 ,

[0111] n 1 ELELVLLTADSLR E21 , A22 , L

[0112] (WT1 ) VEVESNPDLTPEE 23 , L25 , Y2 KEELLRQIDKLRE 9 , A39 , E42

[0113] RARKRLQ , L43 , L46 ,

[0114] T47 , S50 , V

[0115] 53 , E54

[0116] gplO O- A* 02 : 01 YLEPGPV gplO O SLVSEAGKEMARL V3 , G7 , K8 , E29 , I 32 , S36 , T3 TA me 1 ano IRRLPELGEEERR MI O , Al l , I I 39 , N40 , A43 ma LAELKILALSNI I 14 , R15 , L1

[0117] antige NTQAVAGELGQDL 7 , P18 , E29

[0118] n TPELKELYEQIQK , 132 , L33 ,

[0119] TEELPLEEALKRL S36 , 139 , N EEILKELEKKLDT 40 , A43 , V4 LIEQLEQELRE 4 , 193 , LI O

[0120] 0 , R101

[0121] martl- A* 02 : 01 ELAGIGI Melan- DPLKRLTELALEA T7 , A1O , L1 A10 , L14 , Q32 , 3 LTV A / MART LRDEPHVPPEDRP 1 , L14 , P18 N36 , N40 -1 LVTLLQIALNLAI , P22 , R25 ,

[0122] ( 26- NVWNRRHLGRTD P26 , T29 , Q

[0123] 35 ) PEHDRKLLEELEE 32 , 133 , N3

[0124] peptid IRKLPREEAEKRL 6 , 139 , N40

[0125] e EELIERLEEENEK , V43 , H47 ,

[0126] LAEEEVKQFRS E86 , E90 , A

[0127] 93 , V97 , Fl

[0128] 00

[0129] martl- A* 02 : 01 ELAGIGI Melan- SERDRITEAILQF T7 , L11 , A1 A14 , E32 , L33 , 43 LTV A / MART AEQSVEVPESLRR 4 , E15 , V18 T36 , N40 -1 ARTLAELTLTYIQ , R26 , T29 ,

[0130] ( 26- NTIVGLEELGELT E32 , L33 , T

[0131] 35 ) EEQAKEFLKRVEE 36 , N40 , A9

[0132] peptid VIRAL RRGDVERL 3 , L96 , R10

[0133] e EELLKEAEKRWEE 0

[0134]

[0135] VARRLVERRRHprame- A* 02 : 01 ALYVDSL PRAME DEREELVERAVTL V7 , A1O , V1 G36 , S 40 , L44 , A9 FFL (melan TERLGRLGPEERR 1 , T14 , E15 E47 , L93 oma) VAEARIELAGAVL , L17 , G18 ,

[0136] SLAVLREEAGTYD G36 , S40 , V PERFKEDVEAVRK 43 , L44 , E4 LDRLPPEEQLRVA 7 , L93 , T97 EERVRELRKEGVR , R100 , A10 LLEELTRRRAG 1

[0137] prame- A* 02 : 01 ALYVDSL PRAME SRRDRLLEEAIRL L7 , I 11 , T1 E29 , 132 , E33 , A2 FFL (melan TRELPRLGERERF 4 , R15 , L17 L36 , T37 , N40 oma) LAEIRIELLLTEH , P18 , E22 ,

[0138] NLIALSETTGIDF F26 , E29 , I SEHIEKVRKRLEE 32 , E33 , L3 LETLSPEEVRKRA 6 , T37 , N40 PEVEKLLDEVRKA , A43 , T47 , VGRALEEQLRS E82 , R89 , G

[0139] 93 , L96 , L1

[0140] 00

[0141] pap- A* 02 : 01 TLMSAMT Prosta SRLDELLAELDEL L10 , 114 , V V29 , D30 , T33 , 116 NL tic IARRPELPPELFD 29 , D30 , T3 S37 , L40 , L41 , acid VRVDLATTEAS IA 3 , A36 , S 37 E44 , Q63 , Q67 phosph LLEEEIALAERRG , L40 , L41 ,

[0142] atase WEETAAVLRRQLE E44 , L47 , T

[0143] ( PAP ) LQRAQSAVLRARL 56 , V59 , R6

[0144] AALE 2 , Q63 , L66

[0145] , Q67 , Q70

[0146] yfv-2 A* 02 : 01 LLWNGPI Yellow SVELTEEETELLY S 1 , V2 , E8 , A23 , S 46 , V47 ,

[0147] AV fever ESVELLIEAARYD Li l , L12 , E A50 , Y53 vi rus FRYLAKQGFPDEL 14 , S 15 , L1

[0148] (YFV) LRAYHESVKTAFE 8 , L19 , E21

[0149] NS 4b21 YNMEVMTKLPEEK , A22 , A23 ,

[0150] 4-22 KEKLKELLKEAEK Y25 , R28 , Y

[0151] EFEELLH 29 , L39 , A4

[0152] 2 , S46 , V47

[0153] , T49 , A50 ,

[0154] Y53

[0155] yfv- 14 A* 02 : 01 LLWNGPI Yellow GRPYTPEEIQALL R2 , E8 , A11 S 19 , A22 , T23 ,

[0156] AV fever EAAELSLEATKID , L12 , E14 , E46 , T47 , A50 , vi rus IKYLTKQGFSKES A15 , L18 , S Y53 (YFV) LLTIAEETLTAIR 19 , E21 , A2

[0157] NS 4b21 YNAELLKDAPEEV 2 , T23 , 125

[0158] 4-22 QKRMKELEKEAKK , Y29 , T42 ,

[0159] VLEEIKH E45 , E46 , T

[0160] 47 , T49 , A5

[0161] 0 , Y53

[0162] phox2b C* 07 : 02 QYNPIRT Phox2B ARAAEIAALLAAV E21 , L24 , I E21 , L24 , 125 , -10 TF (neuro EADPSTLEDALIA 25 , Q28 , T2 Q28 , E49 , L52 , (RA10_ blasto LQTLALELALAGE 9 , L32 , A35 Y56

[0163] PC ) ma ) ITEEQLLEIERHL , L46 , E49 ,

[0164] NAAYVDLTRDKDR R50 , L52 , N AAALAHLRAAADL 53 , Y56 , V5

[0165] LLAVL 7 , T60 , R61

[0166] phox2b C* 07 : 02 QYNPIRT Phox2B DTVAALRALAARA E21 , 125 , S E21 , 125 , E49 , -7 TF (neuro LADPSHFEDFLIF 28 , T29 , Y3 Y56

[0167] blasto VSTTVYELLAAGR 2 , L35 , A36

[0168] ma ) ITEEEALALEREI , L46 , E49 ,

[0169] NAAYVTRTVTKDR R50 , N53 , Y

[0170] AAL EAHI AAI LAL 56 , V57 , T 6

[0171] FERIE 0 , V61

[0172] phox2b C* 07 : 02 QYNPIRT Phox2B ARAAEIAALLAAV S 18 , E21 , D E21 , L24 , Q28 , -5 TF (neuro EADPSTLEDALIA 22 , L24 , 12 L32 , E49 , L52 , blasto LQTLALELALAGE 5 , Q28 , T29 N53 , Y56 ma ) ITEEQLLEIERHL , L32 , E33 ,

[0173] NAAYVALTRDKDR A35 , L36 , E AAALAHLRAAADL 42 , L45 , L4

[0174] LLAVL 6 , E49 , R50

[0175] , L52 , N53 ,

[0176]

[0177] Y56 , V57 , T60 , R61 , K6

[0178] 3

[0179] phox2b C* 07 : 02 QYNPIRT Phox2B ARLERLRAALAAG E28 , L31 , T E35 , Y42 , Y68 -11 TF (neuro LARLEAARDDLAA 32 , E35 , A3

[0180] blasto LEAELTALEAEVN 6 , V38 , N39

[0181] ma ) ALYVETVAERGAL , A40 , Y42 ,

[0182] SPEEERLINEALL V43 , V46 , G RAYQLLAEALDAA 50 , L52 , S5

[0183] A 3 , P54 , E57

[0184] , R58 , I 60 .

[0185] N61 , L64 , L

[0186] 65 , Y68 , Q6

[0187] 9 , L71 , A72

[0188] , E73 , L75 ,

[0189] D76

[0190] phox2b C* 07 : 02 QYNPIRT Phox2B TAHLITLKVELNG 116 , T17 , T I 16 , T18 , F20 -9 TF (neuro ETITTIFVTLDED 18 , I 19 , F2

[0191] (RA9 P blasto PEELRAYTREAAR 0 , V21 , L30

[0192] C ) ma ) AEAARAEAEGLKV , A37 , E41 ,

[0193] ELTESTVEAA R44 , A62

[0194] HIV_L_ A* 02 : 01 KLTPLCV HIV TVELVQEAAEAVE P58 , Q61 , L L62fI 65fN69f10 TL envelo KLAELLGKEQKGE 62 , K64 , 16 E93 , Q96 pe RTPEWKEREEQL 5 , K68 , N69

[0195] protei KKLEELAKKLENG , L72 , L76 ,

[0196] n DPELQPVAQLLKI E81 , A82 , V VLKNVELMESLGT 83 , L85 , S 8 PSEAVLLSTEAVA 6 , E88 , A89 IELKQEAKKRS , V90 , 192 ,

[0197] E93 , Q96

[0198] HIV_L_ A* 02 : 01 KLTPLCV HIV MRLLEVTVTIKVY Ml , R2 , L3 , D23 , L24 , L25 , 6 TL envelo DENGNNVTEDLLS E5 , E22 , D2 S26 , Y27 , V28 , pe YVASLSEESEEEV 3 , L24 , L25 E33 , L46 , M49 protei IEGVRRLAEMTKA , S26 , Y27 ,

[0199] n LVELYKKQGYKVE V28 , S30 , S VEVEVTVHGGPLP 32 , E33 , E3 EELERAVEEEKEE 4 , E38 , G42

[0200] LKRLQEE , R45 , L46 ,

[0201] M49 , A52 , L

[0202] 53 , L56 , Y5

[0203] 7 , H73

[0204] phox2b C* 07 : 02 QYNPIRT Phox2B DRLARIRALLAAF E21 , L24 , I E21 , L24 , Q49 , -3 TF (neuro AADPAAFEDLLIE 25 , Q28 , T2 Y56

[0205] blasto AQTLILELIEQGV 9 , L32 , 135

[0206] ma ) LTEEEELEAQRLL , L46 , Q49 ,

[0207] NDAYVAHVREGDK R50 , N53 , Y AAALAHFEALIEL 56 , V57 , V 6

[0208] LEKAL 0 , R61

[0209] phox2b C* 07 : 02 QYNPIRT Phox2B AEKEEILKKLKAV E21 , 125 , Q E21 , 125 , E49 , -4 TF (neuro KEDPSNIEDLLIY 28 , T29 , Y3 L52 , Y56 blasto LQTKAYELALAGK 2 , A35 , L46

[0210] ma ) ITEETLLEIEREL , E49 , R50 ,

[0211] NAAYVALTRRKDL L52 , N53 , Y AEALAHIEKAVEL 56 , V57 , T 6

[0212] LLKVL 0 , R61

[0213] phox2b C* 07 : 02 QYNPIRT Phox2B D PVAALAAAAAAA L24 , 125 , S L24 , E49 , Y56 - 6 TF (neuro RADPAAFEDLLIL 28 , T29 , Y3

[0214] blasto ASTLLYEQLAAGR 2 , L35 , L46

[0215] ma ) LTEEEALALEAAL , E49 , A50 ,

[0216] NALYVTRTVTRDE N53 , Y56 , V AAARAHLDRILAL 57 , T 60 , V6

[0217] FRAAA 1

[0218] phox2b C* 07 : 02 QYNPIRT Phox2B SALERIRELLAKF E21 , 124 , I E21 , 124 , E49 , -2 TF (neuro REDPSAFEDLI IA 25 , Q28 , E2 Y56

[0219] blasto LQEQILELIEAGK 9 , L32 , 135

[0220] ma ) LTEEEELELERLL , L46 , E49 ,

[0221]

[0222] NEAYVRHWDGDK R50 , N53 , YEAAIAAFEEAIEL 561V57tV6

[0223] IEKAL 0 , V61

[0224] phox2b C* 07 : 02 QYNPIRT Phox2B DEAAEAAALEAEV L32 , M35 , A M35 , E57 , L60 -8 TF (neuro AALLAQAAADPSR 36 , S39 , Y4

[0225] blasto ADELFLEAMARIS 2 , V43 , Y46

[0226] ma ) AWYVAEYRRHGLL , G50 , L51 ,

[0227] TPEHEARLLALLT P54 , E57 , L AIYDLEAALLDAA 60fL61fL6

[0228] A 4 , Y68 , E71

[0229] , A72 , L75

[0230] phox2b C* 07 : 02 QYNPIRT Phox2B MAKWTLTFRRDG A2 , V4 , R11 T16 , Q17 , Q19 , -12 TF (neuro HVTQIQTVFTPDD , H14 , T16 , T20

[0231] blasto SPAMKAATEAELA Q17 , 118 , Q

[0232] ma ) RIVAEARAAGEEV 19 , T20 , V2

[0233] EVEERESALA l , M30 , A33

[0234] , E37 , I 41 ,

[0235] S59 , L61 , A

[0236] 62

[0237] phox2b C* 07 : 02 QYNPIRT Phox2B RVRLITVTETLNG V2 , L4 , L11 E14 , 116 , T17 , -14 TF (neuro ETITTTFVLTEED , N12 , E14 , T18 , T19 , F20 , blasto APEVRAYVDATVA 116 , T17 , T V21 ma ) AMAAEAKAAGLPV 18 , T19 , F2

[0238] TVEEREVELP 0 , V21 , T37

[0239] , V59 , L61 ,

[0240] P 62

[0241] phox2b C* 07 : 02 QYNPIRT Phox2B SAAQAAAEAAAVA Y22 , T26 , M E44 , 147 , E58 -15 TF (neuro LRRALAATYDEIT 29 , R30 , A3

[0242] blasto ALMRAMAAAGPEE 3 , E44 , 147

[0243] ma ) RRKLETQILLLQT , L55 , E58 ,

[0244] QLLLAEAALLGPE A59 , 169 , E AAPIRAELAAQQA 72 , L73

[0245] ALQAQ

[0246] phox2b C* 07 : 02 QYNPIRT Phox2B SAAFAAQEAARLA L12 , L15 , A E44 , L47 , N51 , -16 TF (neuro ILAEMAPELLAAV 16 , A19 , L2 D58

[0247] blasto AELRAALGGSPEA 2 , V26 , R30

[0248] ma ) RRLAEDRLLLLNL , E44 , L47 ,

[0249] KLIAADEAVLGTD L48 , N51 , I

[0250] AAAMAAE LAAAE A 55 , D58 , L6

[0251] ALAAR 2

[0252] phox2b C* 07 : 02 QYNPIRT Phox2B AETIHISAVLASY 14 , H5 , 16 , H5 , I 6 , S7 , A8 , -20 TF (neuro PYMSDETFRRQLD S7 , A8 , V9 , V9 , L10 , L28 blasto ALVAGVKAAAAAG L10 , Al l , Y

[0253] ma ) YKVDIDLEVHLHP 15 , M16 , T2

[0254] SHASLKPHAEEAK 0 , R23 , Q24 KEIEAILAAQ , A27 , L28 ,

[0255] A30 , G31 , A

[0256] 34 , A35 , A3

[0257] 8 , Y40

[0258] phox2b C* 07 : 02 QYNPIRT Phox2B AETLRIVAVLELT E2 , L4 , R5 , R5 , I 6 , V7 , A8 , -21 TF (neuro PETDPDWLAQLR I 6 , V7 , A8 , V9 , El l blasto AVYDGVASAKAAG V9 , E11 , T1

[0259] ma ) LAVEFDLDVHVHP 3 , E15 , V20

[0260] SREAERARLEAAR , Q24 , A27 , AEAEARVAAA G31 , S34 , A

[0261] 35

[0262] phox2b C* 07 : 02 QYNPIRT Phox2B AALEEELRAALAA S22 , V25 , G E43 , V46 -31 TF (neuro AEEVELAESAAVG 26 , V27 , V3

[0263] blasto VDPEAVAPLVRER 2 , L35 , E38

[0264] ma ) AELEYAVLVAEAA , R39 , L42 ,

[0265] RDPRYREIMRLTL E43 , V46 , A EGLRSLLAAAAAA 49 , R56 , E5

[0266] A 9 , 160 , L63

[0267] , T 64 , G67 ,

[0268] S70 , L71 , A

[0269] 74

[0270] phox2b C* 07 : 02 QYNPIRT Phox2B TVTETLTITFKTT L18 , E21 , R E21 , Y28 , E51 ,

[0271]

[0272] -32 TF (neuro EADILAVEREIND 22 , N25 , Y2 154 , T55blasto RYVEARAAAARLP 8 , V29 , R32

[0273] ma ) PERREAVLARWEA , E51 , A52 ,

[0274] LITELQLQLYTAI 154 , T55 , Q LEALDSGAVGETR 58 , L59 , Y6

[0275] TLTGELTVEL 2 , T63 , 165

[0276] 39 phox2b C* 07 : 02 QYNPIRT Phox2B TVERRAI SPEERR R13 , L16 , L L16 , F19 , V20 , -33 TF (neuro ALLLAFVAAGEPA 17 , F19 , V2 G23 , E24 , P25 , blasto IWTSLGDRATLD 0 , G23 , E24 A26 , V28 , V29 , ma ) AEVAEILAGLRAA , P25 , A26 , T30

[0277] GVEDITI FGVNVA 127 , V28 , V

[0278] TGETVLVRER 29 , T30 , 14

[0279] 5 , G48 , L49

[0280] , A52 , V54 ,

[0281] V62 , V64

[0282] 40 wtl- 8 A* 02 : 01 RMFPNAP Wilms DVEVARHQYELLV 1 , V4 , H7 , Q K66 , N73 , 176 ,

[0283] YL tumor KYLRTQPELRPEV 8 , Li l , Y15 V77 , E80

[0284] antige EEVEKLVERGDLE , L49 , L52 ,

[0285] n 1 ELRKRADEKLDEL 156 , L65 , K

[0286] (WT1 ) EELIKNGGPVEEL 66 , T72 , N7 KRLGELTNLWIVA 3 , 176 , V77

[0287] AEYLETER , E80D

[0288] 56 A* 02 : 01 YLEPGPV gplO O DLVERAGLEMTRL V3 , E4 , G7 ,

[0289] TA IRQLPKLGPEERR L8 , M1O , T1

[0290] LAELRILALSLI I 1 , 114 , R15 NIAGVADELGEDR , L17 , P18 , TEELTKKFEEVKE E29 , 132 , L TQKLPVEEALKKL 33 , S36 , 13

[0291] gplO O- EKILKELEEELDK 9 , N40 , V44 E29 , I 32 , S36 , 68 LIKRLEEERRN , I 93 , R100 139 , N40 57 A* 02 : 01 YLEPGPV gplO O V3 , S 4 , G7 ,

[0292] TA DLVSRAGKRMAEL K8 , M1O , A1

[0293] IKRLPEAGPEERR 1 , 114 , K15 LLELRIENLALI I , L17 , P18 , NVQSVAGELGEDV P22 , 132 , A TEELERKFEEVLK 36 , 139 , N4 TEELPPEEARKRL 0 , S43 , V44

[0294] gplO O- EKLNEELEKELDE , I 93 , A100 114 , 132 , A36 , i37 LIKRLEERARK , R101 139 , N40 , S 43 58 A* 02 : 01 YLEPGPV gplO O V3 , S 4 , G7 ,

[0295] TA DLVSRAGKRMAEL K8 , M1O , A1

[0296] IRKLPELGPEERE 1 , 114 , R15 LAELRILALSLI I , L17 , P18 , NTQAVAEELGEDL 132 , L33 , S TPELSRRFEEVEK 36 , 139 , N4 TEELPPEEARERL 0 , A43 , V44

[0297] gplO O- DRLLDELEKRLDE , L100 , R10 I 14 , I 32 , S36 , T5 LIKRLEERLRS 1 I 39 , N40 , A43 59 A* 02 : 01 YLEPGPV gplO O V3 , E4 , G7 ,

[0298] TA SLVERAGRQMAEL R8 , M1O , A1

[0299] IRRLPELGPEERR 1 , 114 , R15 RAELRILNLSLI I , L17 , P18 , NIAAVAGELGRDI 132 , L33 , S TEELERTFQEVQK 36 , 139 , N4 TEELPLEEALERL 0 , A43 , V44

[0300] gplO O- EELNTRLEKRLDE , L89 , I 93 , I 14 , I 32 , S36 ,

[0301]

[0302] T6 LIKELEERLRS L100 , R101 I 39 , N40 , A43

[0303] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. The interface residues with the relevant pMHC target are shown in the column labeled as “Interface residues” in Table 1. Inone embodiment, at least 5 identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In a further embodiment, at least 10 identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In another embodiment, all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence.

[0304] As used herein, conservative amino acid substitutions involve replacing a residue by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common sidechain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0305] In another embodiment, at least 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, or all identified interface residues are identical (not substituted), relative to the reference sequence.

[0306] In a further embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. The specific residues are shown in the last column of Table 1. These specific residues make direct interactions with peptide residues of the pMHC and are helpful for specific pMHC binding. In one embodiment, at least 3 identified specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In a further embodiment, at least 6 specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In another embodiment, all identified specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all specific residues are identical (not substituted), relative to the reference sequence.In a further embodiment, all interface residues and all specific residues are identical (not substituted), relative to the reference sequence.

[0307] In another embodiment, substitutions relative to the reference sequence are conservative amino acid substitutions.

[0308] In another embodiment, the disclosure provides fusion proteins, comprising:

[0309] (a) the polypeptide of any embodiment or combination of embodiments herein; and

[0310] (b) one or more functional domains at the N-terminus and / or at the C-terminus of the polypeptide.

[0311] In these embodiments, any functional domain may be inserted at one or both termini of the fusion protein. In various non-limiting embodiments, the functional domain may comprise, for example, a detectable domain, a scaffold domain, a secretion signal, one or more components of a chimeric T-antigen construct, one or more components of a bi-specific T-cell engager construct (including but not limited to CH2 and / or CH3 domains from an Fc (fragment, crystallizable) domain, etc. In all embodiments, the polypeptide and one or more functional domains may be directly fused or may be separated by an amino acid linker of any length and amino acid composition as appropriate for an intended use.

[0312] In one embodiment, the fusion protein comprises a chimeric antigen receptor (CAR). CARs typically include an (1) extracellular domain, comprising an antigen binding region and a hinge region that connects the antigen binding region to the antigen binding region; (2) a transmembrane domain capable of anchoring the CAR to T-cell membrane; and (3) an intracellular domain comprising (a) a signaling domain (which may, in some embodiments, comprise a CD3(^ chain), which is capable of transmitting signals to activate the T cell and trigger its cytotoxic function, and (b) one or more costimulatory domains (which may, in some embodiments, comprise CD28 or 4- IBB), which are capable of enhancing T cell activation and persistence. The CAR may comprise other domains, such as a signal peptide, a purification tag, a selectable marker (including but not limited to an antibiotic resistance protein), etc. In the fusion proteins of the present disclosure, the polypeptide of any embodiment or combination of embodiments herein serves as the antigen binding region in the extracellular domain. As a result, the CAR constructs can be used, for example, in CAR-T cells to target the pMHC complex with the polypeptide of the disclosure present in the CAR.

[0313] In one embodiment, the fusion protein comprises the formula X1-X2-X3-X4, whereinXI comprises the polypeptide of embodiment of combination of embodiments herein; X2 comprises a hinge domain;

[0314] X3 comprises a transmembrane domain;

[0315] X4 comprises an intracellular domain;

[0316] optionally including an amino acid linker between 1, 2, or all 3 of X1-X2; X2-X3; and X3-X4.

[0317] Any hinge domain, transmembrane domain, and intracellular domain may be used as appropriate for a CAR and an intended purpose. In one embodiment, one or more of the following is true:

[0318] (i) the hinge domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:43;

[0319] (ii) the transmembrane domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44; and / or

[0320] (iii) the intracellular domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, linked in N- to C-terminal order.

[0321] The amino acid sequences of these exemplary hinge, transmembrane, and intracellular domains are provided in Table 2.

[0322] In a further embodiment, the fusion protein comprises the formula Z1-Z2-Z3, wherein (d) Z1 comprises the amino acid sequence at least 50%, 75%, 90%, or 100% identical to the amino acid sequence of SEQ ID NO: 60;

[0323] (e) Z2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the polypeptide of any embodiment or combination of embodiments herein; and

[0324] (f) Z3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61.

[0325] Table 2. Exemplary CAR construct components

[0326] SEQ ID CAR coiponents and full sequence

[0327]

[0328] NO41 CD28 signal MLRLLLALNLFP S I QVTGGS S

[0329] peptide

[0330] 42 FLAG DYKDDDDK

[0331] 43 45aa_hCD8_hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0332] 44 CD28TM domain VLWVGGVLACYSLLVTVAFI I F

[0333] 45 41bbl VRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL cytoplasmic

[0334] domain

[0335] CD3Z cytoplasmic LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP 46 domain QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR

[0336] 47 P2A ATNFSLLKQAGDVEENPGP

[0337] 48 mTagBFP MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKWEGGPLP FAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDT SLQDGCLI YNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGS HL I ANAKT T YRS KKPAKNLKMP GVYYVD YRLE RI KE ANNE T YVE QHEVAVAR YCDLPSKLGHKLN

[0338] 50 Puromycin MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTR resistance gene VGLD I GKVWVADDGAAVAVWTT PE SVEAGAVFAE I GPRMAELSGSRLAAQQQMEGL LAPHRPKEPAWFLATVGVSPDHQGKGLGSAWLPGVEAAERAGVPAFLETSAPRNL PFYERLGFTVTADVEVPEGPRTWCMTRKPGA NA complete MLRLLLALNLFPSIQVTGGSSDYKDDDDK (SEQ ID NO : 60) -Z2- expression open GTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRPPSKPFW reading frame VLWVGGVLACYSLLVTVAFI I FWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMVSKGEELIKENMHMKLY MEGTVDNHHFKCTSEGEGKPYEGTQTMRIKWEGGPLPFAFDILATSFLYGSKTFI NHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNF TSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTYRSKK PAKNLKMP GVYYVD YRLE RI KE ANNE T YVE QHEVAVAR YC DL P S KLGHKLNGS GAT NFSLLKQAGDVEENPGPMTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDP DRHI ERVTELQELFLTRVGLDI GKVWVADDGAAVAVWTTPESVEAGAVFAEI GPRM AELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAWLPGVEAA ERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA (SEQ ID

[0339]

[0340] NO : 61)

[0341] In another embodiment, the fusion protein comprises a bi-specific T-cell engager of the general formula X1-X2-X3, wherein XI comprises the polypeptide of any embodiment or combination of embodiments herein, X2 comprises an Fc CH2 domain, and X3 comprises an Fc CH3 domain, wherein XI, X2 and X3 can be in any order in the fusion protein.

[0342] Bi-specific T-cell engagers according to this embodiment can be used to activate T cells cytotoxic activity against the pMHC target of the polypeptides of the disclosure. In this embodiment, the construct is capable of forming a dimer with another protein including CH2 and CH3 domains. The other protein also comprises an scFv domain that can engage an immune cell. Any scFv that can engage an immune cell can be used in the second protein as appropriate for an intended use.

[0343] In one embodiment, the domain order is X1-X2-X3. In another embodiment(a) X2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and

[0344] (b) X3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55 APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK AK (SEQ ID NO: 54) GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0345] (SEQ ID NO: 55)

[0346] In a further embodiment, the fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52. In this embodiment, the construct is a dimer so that the fusion protein of SEQ ID NO:52 dimerizes with the polypeptide of SEQ ID NO:53 though the CH2 and CH3 Fc domains found in both polypeptides.

[0347] The disclosure also provides dimers, comprising

[0348] (a) the bi-specific T-cell engager fusion protein of any embodiment or combination of embodiments herein; and

[0349] (b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52 or 53. The amino acid sequences of SEQ ID NO:52-53 are shown in Table 3. In this embodiment, the binder is the polypeptide of SEQ ID NO: 11. The CH2 domain in each chain is in bold font, the CH3 domain is underlined, and the scFv in the SEQ ID NO:53 is in italics

[0350] Table 3. Exemplary bi-specific T-cell engagers.

[0351] MART1_BL

[0352] 13- MGWSCIILFLVATATGVHSDYKDE DPLKRLTELALEALRDEPHVPP hs!gGl_L EDRPLVTLLQIALNLAINVVVNRRHLGRTDPEHDRKLL ALAPG_Kn

[0353] obDS- EELEEIRKLPREEAEKRLEELIERLEEENEKLAEEEVK tev-His- Avi QFRSGGGGSGGGSGGGSGGGGEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM

[0354] ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW

[0355]

[0356] LNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKENLYFQGSHHHHHHGSGLNDIFEAQKIEWHE

[0357] 53 hsCD3_Hi MGWS C 11 L FLVATAT GVH S Q TWTQEPSL TVSPGGTVTL TCRSS TGAVTTSNYANWVQQKP gh_scFv- GQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGG hs!gGl_L TKLTVLGGGGSGGGSGGGSGGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVR ALAPG_Ho QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV leDS

[0358] (High RHGJVFGJVSYVSJVFAYJVGQGTFWVSSGGGSGGGEPKSSDKTHTCPPCPAPEAAGGPSVFLF Affinity PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS CD 3 VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI SKAKGQ P RE PQVCT L P P S RDELT KNQVS L scFv) SCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS

[0359] VMHEALHNHYTQKSLSLSPGK

[0360]

[0361] In one embodiment of the polypeptide or fusion protein of any embodiment herein, the polypeptide binds its target with nanomolar affinity.

[0362] In another embodiment, the disclosure provides kits

[0363] (a) the bi-specific T-cell engager fusion protein of any embodiment or combination of embodiments herein; and

[0364] (b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53.

[0365] In another aspect the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded peptide or chimeric molecular construct, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptide or fusion protein of the disclosure.

[0366] In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence.

[0367] “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The controlsequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.

[0368] In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate coprecipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. In some embodiments, the host cells are T-cells, such as CAR-T cells.

[0369] The disclosure also provides pharmaceutical compositions, comprising:

[0370] (a) the polypeptide, the fusion protein, the nucleic acid, the expression vector, the host cell, the kit, and / or the dimer of any embodiment or combination of embodiments herein; and

[0371] (b) a pharmaceutically acceptable carrier.

[0372] The compositions may be used, for example, in the methods of the disclosure. The compositions may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol,m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, like glycine. In yet other embodiments, the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition additionally includes a stabilizer, e.g., a molecule that substantially prevents or reduces chemical and / or physical instability of the nanostructure, in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0373] The polypeptide, fusion protein, nucleic acid, expression vector, and / or host cell may be the sole active agent in the composition, or the composition may further comprise one or more other agents suitable for an intended use.

[0374] In another aspect, the disclosure provides methods for treating or limiting a disorder associated with a target pMHC in Table 1, comprising administering to a subject in need thereof an amount effective to treat or limit development of the disorder of the polypeptide, fusion protein, nucleic acid, expression vector, cell, dimer, or pharmaceutical composition of any embodiment or combination of embodiments herein.

[0375] As noted above, a polypeptide, fusion protein, nucleic acid, expression vector, cell, or pharmaceutical composition of any embodiment of the disclosure may be used, for example, in protein and cell based pMHC targeting to treat disease associated with the target pMHC. By way of non-limiting example, the target pMHC of the polypeptides of SEQ ID NO:4-5 is a pMHC associated with SAR-CoV-1 infection, and thus can be used to treat or limit development of a SARS-CoV-1 infection; the target pMHC of the polypeptides of SEQ ID NO:6-7 is a pMHC associated with HIV infection, and thus can be used to treat or limit development of an HIV infection; and the target pMHC of the polypeptides of SEQ ID NO: 8-9 is a pMHC associated with Wilms tumor protein, and thus can be used to treat or limit development of malignancies associated with Wilms tumor protein such as leukemia and various solid tumors.As used herein, "treat" or "treating" a disorder means accomplishing one or more of the following in a subject with the disorder: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

[0376] As used herein, “limiting development” of a disorder means administering to a subject that does not have the disorder but who is or may be at risk of developing the disorder, to limit or prevent development of the disorder and its symptoms.

[0377] The subject may be any subject that has a relevant disorder or may be at risk of the relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. In a specific embodiment, the subject is a human subject.

[0378] As used herein, an “effective” amount refers to an amount of the polypeptide, fusion protein, nucleic acid, expression vector, host cell, and / or pharmaceutical composition that is effective for treating or limiting development of the disorder. The polypeptides, fusion proteins nucleic acids, expression vectors, and / or host cells are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including but not limited to orally, by inhalation spray, ocularly, intravenously, subcutaneously, intraperitoneally, and intravesicularly in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0379] Any suitable dosage range may be used as determined by attending medical personnel. Dosage regimens can be adjusted to provide the optimum desired response. A suitable dosage range for the polypeptides or fusion proteins may, for instance, be 0.1 ug / kg-100 mg / kg body weight; alternatively, it may be 0.5 ug / kg to 50 mg / kg; 1 ug / kg to 25 mg / kg, or 5 ug / kg to 10 mg / kg body weight. In some embodiments, the recommended dose could be lower than 0.1 mcg / kg, especially if administered locally (such as by intra-tumoral injection). In other embodiments, the recommended dose could be based on weight / m2 (i.e. body surface area), and / or it could be administered at a fixed dose (e.g., .05-100 mg). The polypeptides, fusion proteins, nucleic acids, expression vectors, and / or host cells can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.Examples

[0380] Class I MHC molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance, and specific targeting of these peptide-MHC (pMHC) complexes could have considerable utility for treating diseases. Such targeting is challenging as it requires readout of the few outward facing peptide antigen residues and the avoidance of extensive contacts with the MHC carrier which is present on almost all cells. Here we describe the use of deep learning-based protein design tools to de novo design small proteins that arc above the peptide binding groove of pMHC complexes and make extensive contacts with the peptide. We identify specific binders for ten target pMHCs which when displayed on yeast bind the on-target pMHC tetramer but not closely related peptides. For five targets, incorporation of designs into chimeric antigen receptors leads to T-cell activation by the cognate pMHC complexes well above the background from complexes with peptides derived from proteome. Our approach can generate high specificity binders starting from either experimental or predicted structures of the target pMHC complexes and should be widely useful for both protein and cell based pMHC targeting.

[0381] We reasoned that de novo protein design could provide a powerful approach to the specific pMHC recognition problem. We reasoned that if such binders could be generated against pMHCs there could be multiple advantages in stability, engineerability, and manufacturability, and set out to explore the computational design of high affinity and specificity pMHC binders.

[0382] Results:

[0383] Critical to pMHC recognition for therapeutic applications is for binding to be dependent on the identity of the peptide, as MHCs can present other peptides from the proteome that share similar sequences (Fig. la); indeed, high specificity TCRs and scFvs make extensive interactions with the presented peptides (3, 5). To specifically target the peptide in pMHC complexes, we used the generative Al protein design method

[0384] RF diffusion™ to generate protein backbones specifying the upward facing (out of the MHC groove) residues of the peptide as hotspots (Fig.lb). Starting from random Gaussian distributions of amino acid residues placed adjacent to the peptide, diffusive denoising trajectories generated a wide variety of small protein backbones that arc above the central peptide binding groove of the MHC (Fig.2a). From tens of thousands of independent trajectories, we selected backbones capable of hosting side chains making many contacts with the peptide based on contact molecular surface (CMS) (77) (Fig.lb). Following sequencedesign with ProteinMPNN™, we selected for further consideration the designs which AlphaFol d2™ (AF2) (72) predicted to fold and bind as designed. Once such designs were identified for the first few targets, we explored using partial diffusion (73) to adapt the most promising scaffolds to new pMHC targets (Fig. 1c). Starting from a shape complementary binder to one member of a protein family, partial diffusion can rapidly generate high specificity binders to other family members (6); this partial diffusion based customization of promising scaffolds for new targets could reduce computational cost as only a small fraction of de novo RF diffusion™ trajectories generate backbones which make extensive contacts with the peptide while largely avoiding the MHC.

[0385] For pMHC recognition for therapeutic applications, specificity is of the utmost importance. We used two computational approaches to evaluate the specificity of the RF diffusion™ / ProteinMPNN™ generated binders for the intended target peptide. First, we used ProteinMPNN™ to evaluate the effect of mutating each amino acid of the target peptide for each designed protein-pMHC complex. We selected designs for which the peptide amino acid had high probability at each position, and for which substitutions at each position reduced this probability considerably (Fig.2c). Second, for each target, we identified closely-related peptides (see methods) in the human proteome that are presented by the target HLA allele. We used a finetuned AF2 model to predict the structure of each design with both the targeted and off-target peptides in complex with the MHC, and selected designs predicted to bind the on-target peptide (lower pAE scores) considerably more confidently than the off-target peptides (higher pAE scores) (Fig.2d).

[0386] To evaluate the ability of this design pipeline to generate binders to a wide range of pMHC complexes, we selected a structurally diverse set of ten complexes comprising HLA alleles A*01:01, A*02:01 and A*03:01 presenting 9- and 10-mer peptides from viral proteins, tumor associated proteins, and neoantigens. For each target, we obtained oligonucleotide pools encoding 200-12,000 designs, displayed the designs on yeast, and selected those that specifically recognized the targeted pMHC, but not 2-4 closely related peptides (Table 4) on the same MHC (referred to off-target peptides below), by dual color fluorescent cell sorting (FACS) (Fig.4a). Top designs were selected through NGS enrichment analysis or clonal selection (Fig.3a). For seven of the ten pMHC complexes, we used de novo diffusion, and identified designs with a range of topologies (Fig.3a, left) and peptide binding interfaces (Fig.3a, right) that specifically bound the target peptide, with reduced binding to off-target peptides on the same HLA (Fig.3b). For three of the targets, we used the partial diffusion approach starting from one of the de novo designs (Fig.3c top); we again identifiedspecific on-target binders in each case (Fig.3c down). While we were not able to solve structures experimentally of the designs in complex with pMHC, AF3 and Chai-1 14, 15) predictions of the structures of the designed proteins in complex with pMHC were very similar to the design models (Table 4, Fig.4b and c, right; for simplicity we refer to these interchangeably as the design model below). We describe the results for each class of targets in the following paragraphs.

[0387] Allele Target On-target Off-target peptides (genes)

[0388] peptide

[0389] \ A*01:01 | MAGE-A3 | EVDPIGHLY | ESDPIVAQY (TTN) (SEQ ID \

[0390] | (SEQ ID | NO: 63) \

[0391] | NO:62) \

[0392] § A*02:01 | HIV-Env | KLTPLCVTL | KLQELCCTL (YES1) (SEQ i

[0393] | (SEQ ID | ID NO: 65) i

[0394] | NO:64) | KLNPVCCEL (ATG2B) (SEQ i

[0395] | ID NO: 66) i

[0396] | KLIPLCHQL (CTNNAL1) i

[0397] 1 (SEQ ID NO: 67) |

[0398] § A*02:01 | Wilms tumor antigen | RMFPNAPYL s RLFPNLPEL (ARHGEF11) i | | 1 | (SEQ ID | (SEQ ID NO:69) i

[0399] I NO:68) | HLYPNTPYA (APEX1) (SEQ i

[0400] | ID NO:70) i

[0401] | RMFPTPPSL (MED13L) I

[0402] | (SEQ ID NO:71) \

[0403] | A*02:01 | SARS-CoV GLMWLSYFV GLAWLSLFV (KCNK5) \

[0404] § membrane protein i (SEQ ID s (SEQ ID NO:73) :

[0405] | NO:72) | GLIFSSYFV (KCNQ1) (SEQ i

[0406] | ID NO:74) i

[0407] | SLMYLSYLV (ELOVL6) i

[0408] | (SEQ ID NO:75) \

[0409] | A*02:01 | YFVNS4b214-22 | LLWNGPIAV | IVWNGPVGV (PGK2) (SEQ \

[0410] | (SEQ ID | ID NO:77) I

[0411] | NO:76) | TLWRGPVW (STEAP2) I

[0412] | (SEQ ID NO:78) i

[0413] | A*( 12:01 | MART-1 | ELAGIGIL TV j GLMGAGIAQV (HADHA) \

[0414] | (SEQ ID | (SEQ ID NO:80) \

[0415] | NO:79) | SLAGLGLWLL (LY6G6C) \

[0416] | (SEQ ID NO:81) \

[0417] | A*02:01 | gplOO \ YLEPGPVTA FQDPVPLTV (TRIM24) \

[0418] I (SEQ ID | (SEQ ID NO:83) \

[0419] I NO:82) | YLQPWPVDV (ASB6) (SEQ \

[0420] | ID NO:84) \

[0421]

[0422] A*02:01 PAP TLMSAMTNL TLMSMVANL (PSMB3)

[0423] (SEQ ID (SEQ ID NO: 86)

[0424] NO:85) TLFSALTGL (ZMPSTE24)

[0425] (SEQ ID NO:87)

[0426] A*02:01 PRAME ALYVDSLFFL ILYVDPLPMI (ATP2A1)

[0427] (SEQ ID (SEQ ID NO:89)

[0428] NO:88) MLTLNSIFYV (ABCA5)

[0429] (SEQ ID NO:90)

[0430] LMYLGSLAFL (ADM) (SEQ

[0431] ID N0:91)

[0432] YLSIDSALFV (PQLC1)

[0433] (SEQ ID NO:92)

[0434] A*03:01 CTNNB1 (S5F) TTAPFLSGK TTAPSLSGK (CNTTB1)

[0435] (SEQ ID (SEQ ID NO:94)

[0436] NO:93)

[0437]

[0438] Table 4: Peptide-MHC targets and selected off-target peptides. pMHC targets with respective HLA alleles, target peptides, and closely related peptides from the human proteome chosen as off-targets (gene of origin indicated in parentheses).

[0439] To evaluate the ability of the design pipeline to generate binders to viral peptide-HLA complexes, we chose three previously characterized epitopes from different pathogens presented on A*02:01: SARS-CoVl / GLMWLSYFV (SEQ ID NO:72) (76), YFV / LLWNGPIAV (SEQ ID NO:76) (77), HIV / KLTPLCVTL (SEQ ID NO:64) (75). For all three targets, we identified designs that distinguish the target peptides from closely related off-target peptides (Fig.3b). To assess whether the designs can maintain the specificity as the binding domain for chimeric antigen receptors (CARs), we made CAR constructs (79) and expressed them on the surface of Jurkat cells. While not all of the CARs expressed, top specific hits that expressed have specific pMHC binding profiles (Fig.3b). In the design models (Fig.3a), the binders make extensive contacts with the target peptide (Table 5). As with TCR-pMHC complexes, target sequence specificity arises in two ways: first by direct interactions between designed side chains and target peptide side chains, and second, by specific recognition of the conformation of the bound peptide by hydrogen bonds with the target peptide backbone, which is also determined by the peptide amino acid sequence. For example, the SARS design buries the target peptide W4, L5 and Y7 in a hydrophobic pocket, and makes flanking hydrogen bonds with the peptide backbones; the YFV design makes hydrogen bonds with N4 and hydrophobic interactions with 17 of the peptide; and the HIV design makes hydrogen bonds with KI, T8, and the peptide backbone. We next evaluated theability of our design pipeline to target diverse tumor associated antigens (TAAs): WT1 (RMFPNAPYL / A*02:01; SEQ ID NO:68) (20, 21), PAP (TLMSAMTNL / A*02:01; SEQ ID NO:85) (22), and the neoantigen CTNNbl (TTAPFLSGK / A*03:01; SEQ ID NO:93 with S5F mutation) (23). We identified specific designs for all three peptides (Fig.3b); as found for the viral antigens, the design models show extensive interaction between peptides and designed binders (Fig.3a). The CTNNbl design makes extensive contact with the mutated Phe residue, through cation-pi, pi-pi, and other hydrophobic interactions (Fig.3a), likely contributing to the specific binding observed for the S5F peptide but not the Wild-Type (WT) CTNNbl peptide (Fig.3b).

[0440] Design:Pre Interface hydrop Interface_polar_s i mhc sasa peptide sas diction Binder hobic sasa (A2) asa (A2) i (A2) a (A2) RMSD (A) YFV 148 2114 892 2692 777 0.822

[0441] SARS 137 1516 716 1746 756 0.811

[0442] HIV 10 1772 934 2359 788 0.933

[0443] WT1 8 1545 1044 2248 761 0.716 WTl_6rsy_tcr 1303 1018 1960 769 NA

[0444] PAP 116 1526 958 : 2249 580 0.454

[0445] CTNNbl_15 1342 579 1729 471 0.463

[0446] MAGE 513 1969 861 2488 793 0.745 MAGE_5brz_tc

[0447] r 1114 766 1586 581 NA

[0448] gp!00_T3 1739 925 2356 719 0.63

[0449] Martl_5_5 1895 972 : 2584 826 0.754 Martl_5nht_tcr 1231 626 1584 764 NA

[0450]

[0451] PRAME A9 1998 807 2300 880 0.555

[0452] Table 5. Design Model Characteristics. Solvent Accessible Surface Area (SASA) values in A2for polar residues and hydrophobic residues at the interface for each target / binder pair, compared to TCR values when available. Delta values for SASA of MHC only and Peptide only are shown with MHC minus Peptide. RMSD of the design model to the Chai-1 or AF3 structure.To test the partial diffusion approach, we started from a design that binds the MAGE-A3 peptide (A*01 :01-EVDPIGHLY; SEQ ID NO:62) (24, 25) but not the off-target Titin peptide (A*01:01-ESDPIVAQY; SEQ ID NO:63) (26) (Fig.3c, top). Starting from this scaffold, we generated specific binders for other 9- and 10-residue tumor associated antigens presented by A*02:01 : the gplOO peptide YLEPGPVTA (SEQ ID NO:82) (27) and the Mart-1 peptide (A2L) ELAGIGILTV (SEQ ID NO:79) (28). As only a small fraction of potential pMHC targets have experimentally determined structures, we also explored the possibility of using predicted pMHC structures as targets for binder design. The PRAME protein is highly expressed in multiple types of tumors, and a peptide derived from PRAME (ALYVDSLFFL; SEQ ID NO:88) (29) is displayed on HLA-A*02:01. Despite the strong therapeutic relevance and wide patient population coverage, no high-resolution structure has been determined. AF3 predictions of the complex are confident but display some flexibility in the central region of the peptide, consistent with the intrinsic flexibility of 10-mer peptides in the MHC. For design, we experimented with using 5 AF3 predicted structures as starting points. For all three peptide-MHC targets (gplOO, Mart-1, and PRAME), we identified specific binders that make extensive and diverse hydrophobic and hydrogen bonding interactions with the target peptide (Fig.3c).

[0453] To evaluate the behavior of our designs as soluble proteins, we expressed the HIV and MAGE- A3 binder designs shown in Fig.3 in A. coli and purified them using nickel-NTA chromatography (Fig.4b and c). The purified designs eluted as single peaks in size exclusion chromatography around expected size. We measured the binding of the designs to their cognate pMHCs using surface plasmon resonance experiments and found the binding affinities to be in the single to double digit nanomolar range (Fig.4b and c).

[0454] We next evaluated the ability of the designs to enable specific T-cell activation by the target pMHC when incorporated into chimeric antigen receptors. To be effective and safe in cell therapy settings, such CARs incorporating designed proteins must mediate specific activation by the target peptide loaded on the target HLA, but not by the thousands of other peptides from the proteome loaded on the same HLA, or by peptides on different HL As. To evaluate such activation specificity, we incorporated the binders into CARs and expressed them on Jurkat cells, incubating them with 293T cells that were treated with on-target or off-target peptides. 293T cells have an intact antigen presentation system and hence present a wide range of peptides derived from self proteins on their surfaces; requiring selective activation by pulsing peptides in this system is thus more stringent than using cells that cannot present self peptides due to defects in presentation.Among the designed MAGE binders, the MAGE-513 design resulted in strong and specific activation only by MAGE- A3 peptide stimulation but not by the off-target Titin peptide, or peptides derived from the intracellular proteome on HLA-A*01:01 (Fig.5a).

[0455] Despite binding to target tetramer specifically (Fig.6a), Other designs had either weak signaling (Fig.6b, MAGE-282) or background activation (Fig.6b, hit-4) likely due to responses to other peptides from the proteome loaded on the same HLA as only HLA-A*01:01 expressing 293T cells give the background activation. In the design model (Fig.5b), MAGE-513 engages the MAGE peptide through hydrogen bonds with the side chain of H7 and backbone of L8 (Fig.5b), and through hydrophobic interactions (mediated by design L37 and L86) with the sidechain of peptide L8 (there are also hydrogen bonds between R33 of the design and N66 of the HLA) (Fig.5b). D29 of the design is close to P4 of the peptide without making a clear interaction (Fig.5b); this residue likely plays a gatekeeper role that contributes to binding specificity by clashing with off target peptides that contain bulky residues at site 4. Consistent with the design model and predicted structure, alanine mutations of the most extensively interacting residues on the peptide (15, H7, L8) disrupt signaling (Fig.5c).

[0456] Similarly, CARs with alanine mutations in designed binder residues (L37, D40, L86) which interact most closely with the peptides show reduced activation compared to WT MAGE-513 upon stimulation (Fig.5d). Mutation of D29 does not influence signal upon pulsing with MAGE peptide but does have higher background signal when co-incubated with HLA-A*01:01 expressing 293T cells likely due to increased cross-reactivity, consistent with a gate-keeper role (Fig.5d).

[0457] Comprehensive understanding of the pMHC binder activation landscape requires library-wide scanning of diverse peptides 30). To further characterize the specificity of the MAGE-A3 design, we used a comprehensive library of peptide-HLA-A*01:01 complexes displayed on yeast. Two of the top three activating peptides found probing this library with the MAGE- A3 design, and then measuring activation of MAGE- A3 CAR T cells by the identified peptides, have similar outward facing sidechains as the MAGE-A3 peptide (Fig.5e). Thus, similar to TCRs, peptides that cross-react and activate signaling through our de novo binders have similar sequences, indicating that our design pipeline can generate specificity for a very small subset of possible sequences within a predictable range.

[0458] Following up on this observation, we carried out a second scan of cross-activating peptides in the human proteome based on sequence similarity 31). The most activating peptides (EVDPIGHVY (SEQ ID NO: 95) and VTDFISHLF (SEQ ID NO: 96)) again are among themost similar sharing outward facing residues 15, H7, L8 in sequence (Fig.5f), consistent with the alanine scanning results.

[0459] Given these promising results with our designs against the MAGE pMHC, we explored activation of signaling by cells expressing CARs incorporating designs for additional targets. We observed selective activation of signaling for CARs containing designs against gplOO, WT1, MART-1, and HIV antigens (Fig.8a-e, Fig.7a, compare “Target peptide” to “DMSO” histograms). For WT1, gplOO, and MART-1, pulsing 293T cells with the targeted peptide activated at the same or higher levels than pulsing with almost all single alanine peptide variants (Fig8.a-e; for HIV an alanine mutant increased activation (Fig.7e), with activation patterns consistent with the design models (Fig. 3a). In the design model of gpl00_T3 (Fig. 3c), there are multiple hydrogen bonds with main chains of P6 and V7, which are positioned by interactions between the intervening residues and the pMHC, and thus activation is sensitive to mutations from P4 to V7. For WT1 5, E40 of the binder makes bidentate interactions with Rl, and the main chain of binder 112 makes a hydrogen bond with the side chain of peptide Y8, again consistent with the alanine scanning results. For the MART-1 target, we carried out alanine scanning experiments for two different binders (Mart-1 3 and Mart-1 43; Fig 8.d and e) which are centered over different regions of the target peptide; this structural shift is reflected in the alanine scanning results.

[0460] For PRAME where we used predicted structures rather than experimental structures in the design calculations, we identified two binders (PRAME A9, PRAME A2) that specifically induce Jurkat activation upon PRAME peptide pulsing (Fig.8f). In SPR experiments, the purified designs have KD’S for the target pMHC of 96nM (Fig.8g) and 35nM (Fig.7b). Pulsing 293T cells with the original PRAME peptide and single alanine mutant version showed that binder A9 was quite specific (Fig.8h). In the design model / predicted structure (Fig.8c), the binder interacts with the side chains of V4, S6, L7, F9 and the main chain of L7, consistent with the observed specificity.

[0461] For PRAME A2 (Fig.7c) and the HIV design (Fig.3a), one of the alanine substitutions activated more strongly than the unmutated target peptide (Fig.7d and e). This could reflect better loading of the mutant peptide onto MHC (32), or an imperfect designed interface around the residue being mutated (Fig.7c). Because we have structure models of all designs, the latter problem can potentially be remedied by another round of structure based design. We tested this for the PRAME A2 design and indeed found that activation by the target peptide over the mutant peptide could be increased by redesigning the contacting part of the design to remove a potential clash with the original peptide residue (Fig.7d). Thisability to design and optimize binders to pMHC targets without experimental structures should enable the application of our design pipeline to a very wide range of targets.

[0462] Discussion

[0463] Given the promise of therapies targeting pMHC-I antigens, multiple approaches have been developed to identify specific binders. The central challenge is how to achieve effective targeting of the target pMHC while retaining high specificity — binding to the target peptide but not thousands of other peptides presented on the same MHC. Our de novo design approach generates binders with pMHC interaction interfaces focused on the presented peptide with limited contact with the MHC. We demonstrate that this enables robust design of specific binders for ten diverse pMHC targets; because of the simplicity of the structures (compared to scFvs and TCRs)( Fig.7c and d). Our design pipeline is quite efficient and could be readily applicable to a wide range of pMHC targets; for example in the cases of Mart-1 and gplOO, computational design took one week, and once an oligonucleotide library encoding the designs had been obtained, it took one week to identify specific binders on yeast, and two weeks to subselect specifically activating binders on Jurkat cells.

[0464] We expect the power of our design approach to rapidly design specific binders to class I pMHCs to continue to increase. First, it should be possible to learn from our design campaigns what properties correlate with specific activation on cells; for example, what types of scaffold geometries give the most effective readouts of sequence over the full peptide length. Second, as deep learning-based structure prediction, design, and model ranking methods continue to increase, it should become possible to find designs with suitable affinities and specificities in testing only a small handful of candidates.

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[0528] Methods:

[0529] Computational design of pMHC binders

[0530] Target structures used as inputs for binder design were obtained from the Protein Data Bank for A*01:01 MAGE-A3 (PDB: 5BRZ), A*03:01 CTNNbl (PDB: 6O9C), A*02:01 HIV-Env (PDB: 2X40, AF3 models), A*02:01 Wilms tumor antigen 1 (PDB: 6RSY, AF3 models), A*02:01 SARS-CoV membrane protein (PDB: 3I6G), A*02:01 YFV NS4b214-22 (PDB: 6SS8), A*02:01 MART-1 (PDB: 5NHT), A*02:01 gplOO (PDB: 5EU3). The structure for A*02:01 PAP (TLMSAMTNL; SEQ ID NO:85) was predicted by folding with a version of AlphaFold2™ fine-tuned for MHC (33) structures; the structure for A*02:01 PRAME (ALYVDSLFFL; SEQ ID NO:88) was predicted by folding with AlphaFold3 using all 5model outputs. Upward-facing residues of the peptide were chosen as hotspots to condition RF diffusion™ towards generating binders with high peptide contact. For scaffold recycling, a partial t between 12 and 25 (out of 50 total RF diffusion™ denoising steps) was used after docking scaffolds to new targets. ProteinMPNN™ was used to generate sequences for the output backbones. A range of 1,000-20,000 backbones were generated per RF diffusion™ cycle, with anywhere between 4-32 MPNN sequences generated per backbone. Output structures were subject to in silico screening based on AlphaFol d2™ initial guess (pAE interaction, binder pLDDT and binder RMSD), AlphaFol d2™ monomer pLDDT, Rosetta™ contact molecular surface (CMS) per target residue or range of residues, and ProteinMPNN™ log probability scores of designs in complex with alanine scan mutants of the target peptide. The cutoff values varied for each target protein and round of design. This design process was iterated for each target through new rounds of partial RFdiffusion™ until a desired number of designs passed the cutoffs.

[0531] AF-MHC screening

[0532] Building off previous work in predicting pMHC complexes, prior to the release of AF3, we altered the AlphaFol d2™ fine-tuned for MHC 33) to predict minibinder-pMHC complexes through additional templating of the minibinder. For screening, designs were predicted against the on-target and 2-3 relevant off-target peptides in the same HLA allele. The pAE interaction between only the peptide and the minibinder was calculated, and for each off-target a delta pAE interaction (on-target pAE - off-target pAE, more negative is better) was used to filter designs, with scores ranging from -10 to -0.5 depending on the on-target / off-target pair.

[0533] Rosetta™ CMS screening

[0534] Rosetta™ contact molecular surface described before (77) uses a triangulation algorithm to calculate the contact surfaces of binder and target and gives it a score taking into account cavities and holes in the interface. For peptide contact screening, we restricted the target surface being calculated by the algorithm to the peptide only or to individual residues of the peptide and scored its contact to the entire binder surface. Cutoff values were determined empirically for each target.

[0535] ProteinMPNN™ log probability screeningProteinMPNN™ was used to score the likelihood of each peptide residue being predicted when in complex with a binder. Binders were screened by scoring each peptide residue separately and filtering directly on the log probability of the on-target residue, or on the difference in log probabilities to when that residue is mutated to an alanine.

[0536] Off-target peptide identification

[0537] To determine peptides that are likely to cross react with our peptide of interest, we used selective Cross-Reactive Antigen Presentation (7), and took all the outputs and crosschecked them with NetMHC4.1 (34), using any peptides with an EL rank <= 0.5. Then we used a simple blossom alignment of the passing peptides against our peptide of interest to rank and choose the top off-target peptides.

[0538] DNA library preparation

[0539] For yeast surface display, all designed protein sequences were first padded at both termini using GS to a uniform length of either 88 amino acids or 102 amino acids depending on if a 300bp or 350bp oligonucleotide library was ordered, respectively. Then the sequences were reverse translated using dnachisel™ while codon optimizing for S. cerevisiae. DNA sequences were ordered as oligonucleotide libraries via Twist Biosciences or Integrated DNA Technologies (IDT), with 300bp or 350bp sizes.

[0540] Yeast surface display screening with FACS

[0541] Transformed S. cerevisiae EBY100 strain library cultures were grown in C-Trp-Ura (2% glucose w / v) medium and induced for expression in SGCAA (0.2% glucose w / v) medium. Cells were washed with PBSF (PBS with 1% BSA w / v) and incubated with FITC-conjugated anti-C-Myc chicken antibody (ICL, CMYC-45F) for expression sorting. For binding sorts, cells were additionally incubated with peptide-MHC biotinylated tetramer (Fred Hutchinson Cancer Center Immune Monitoring Services) or dextramer (Immudex) conjugated with phycoerythrin (PE) for on-target peptide or allophycocyanin (APC) for off-target peptide binding. When tetramers or dextramers were mixed, biotin was added to a final concentration of luM. Cells were incubated for 30 minutes, then washed and resuspended with PBSF. All sorts were performed on the Sony SH800 FACS instrument. FACS data was analyzed using the FlowJo™ software. All naive and sorted pools were sequenced using Illumina NextSeq™ and MiSeq™ and analyzed for enrichment between sorts.Protein binder expression and purification

[0542] Synthetic genes of top yeast display hits were ordered as eBlock™ gene fragments from IDT and cloned into a pET29b(+) vector containing an C-terminus His6x tag for all designs and an Avi-tag or SNAC-tag for some designs to introduce Trp to expressed sequences for protein quantification using A280 signal. Following cloning and transformation as in (35), we picked single colonies for sequence verification. Protein purification was similar to (35); briefly, we grew cultures in 50mL of Terrific Broth II with 50mg / mL of kanamycin for 6-12 hours before spiking in IPTG at ImM final concentration and growing overnight at 18C. Cultures were harvested by spinning at >4000g for 5-10 minutes, and resuspending in lysis buffer (15mL of 25mM Tris-HCl, 300mM NaCl, 40mM Imidazole), with addition of protease inhibitor, lysozyme, and DNAse. Following lysis by sonication and centrifuge at > 14,000g for 45 minutes, proteins were purified via nickel Immobilized Metal Affinity Column. Supernatant was allowed to freely drip before washing with 5mL of lysis buffer twice. Proteins were eluted with l-2mL of elution buffer (25mM Tris-HCl, 300mM NaCl, 500mM Imidazole) before SEC using Superdex™ 75 10 / 300GL columns in HBS-EP+ buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, Cytiva #BR100669) and collecting relevant elution fractions.

[0543] Surface Plasmon Resonance

[0544] Binding kinetics were analyzed via Surface Plasmon Resonance (SPR) on a Biacore™ 8K (Cytiva). Binding for different pMHC targets was measured by capturing biotinylated peptide-MHC monomer (Fred Hutchinson Cancer Center Immune Monitoring Services) using the Biotin CAPture™ Kit (Cytiva #28920234). Capture was performed by injecting 0.5 pg / mL pMHC monomer at a flow rate of 10 pL / min in HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, Cytiva #BR100669) aiming for a capture level of -250 response units. Binder analytes in HBS-EP+ buffer were injected at a flow rate of 30 pL / min to monitor association. HBS-EP+ was also used as a running buffer during dissociation under the same flow rate conditions. Ligand concentrations ranged from InM to luM. Binding kinetics were determined by global fitting of curves to konand k0ff assuming a 1 : 1 Langmuir interaction, using the Cytiva evaluation software.

[0545] Chimeric Antigen Receptor constructsCAR plasmids are constructed based on pSLCAR-CD19-BBZ (Addgene: 135992). In short, the configuration from N-terminal to C-terminal is CD28 signal peptide-FLAG-binder-CD8 hinge-CD28 transmembrane domain-4 Ibbl cytoplasmic domain-CD3Z cytoplasmic domain-P2 A-mT agBFP2-P2 A-PuroR.

[0546] Mammalian cell culture and transfection

[0547] Binder sequences were reverse translated while optimizing for H. sapiens and ordered as CAR plasmids from Genscript based on pSLCAR-CD19-BBZ (Addgene: 135992).

[0548] Lentivirus particles of the CAR plasmids were generated by transfecting 0.25mL HEK 293T cells at 800,000 cell / mL grown in DMEM medium (Gibco, 11965092) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (Pen-Strep), with 2 pl TransIT-293 (Minis), 0.25 pg psPAX2 (Addgene, 12260), 0.1 pg pCMV-VSV-G (Addgene, 8454), and 0.4 pg plasmid in with 50 pl OptiMEM™ medium (Gibco, 31985070). Transfected HEK293T cells were replenished with fresh RPMI 1640 medium (Gibco, 61870036) supplemented with 10% (v / v) fetal bovine serum FBS and 1% (v / v) Pen-Strep 24 hours after transfection. Supernatant was collected 48 hours post-transfection and freeze-thawed to kill remaining HEK293T cells. To generate Jurkat-CAR cells, 0.25mL Jurkat cells (a gift from Phil Greenberg lab) at 2 million cells / mL grown in RPMI medium (10% FBS, 1% Pen-Strep) were supplemented with 8 pg / ml Protamine (Millipore™ Sigma, P4020) and infected with 0.4mL of collected lentivirus supernatant by centrifuging at 1,000g for 60 minutes. Lentivirus-infected Jurkat cells were replenished with fresh RPMI growth medium 24 hours post-infection and every 48 to 72 hours thereafter.

[0549] Antigen presenting cell lines

[0550] HLA-A*01:01-expressing HEK 293T were made with lentiviral plasmid expressing human HLA-A*01:01 (a gift from Paul Thomas lab); HEK 293T constitutively expressing HLA-A*02:01 were used as HLA-A2 antigen presenting cells. HLA-A*03:01-expressing K-562 cells were made using lentiviral plasmid expression (a gift from Paul Thomas lab).

[0551] Yeast surface display clonal binding assay

[0552] Clonal yeast surface display samples were obtained by plating sorted pools from FACS on glucose-Trp-Ura agar plates (Teknova C3260), incubating at 30C for 48 hours and picking single colonies. Cultures were grown in C-Trp-Ura (2% glucose w / v) medium and induced for expression in SGCAA (0.2% glucose w / v) medium for 12-18 hours. 50uL ofculture was transferred to a 96-well plate and washed with PBSF. Cells were stained at 1 : 100 v / v with FITC-conjugated anti-C-Myc chicken antibody (ICL, CMYC-45F) for expression and with 1 : 100 v / v peptide-MHC biotinylated tetramer (Fred Hutchinson Cancer Center Immune Monitoring Services) for both on-target peptide (PE-conjugated) and off-target peptide (APC-conjugated). Cells were incubated for 30 minutes and then washed and resuspended in PBSF. All flow cytometry experiments were performed on the Attune NxT instrument and data was analyzed using the FlowJo™ software.

[0553] Jurkat-CAR binding assay

[0554] 50 uL of Jurkat-CAR cells at 1 million cells / mL were added to a 96-well plate.

[0555] Growth medium was removed and replaced with 50 uL staining reagent made of FACS buffer (Dubecco’s PBS pH 7.2, 0.5% bovine serum albumin, and 2 mM EDTA) containing 1 : 100 v / v peptide-MHC biotinylated tetramer (Fred Hutchinson Cancer Center Immune Monitoring Services) for both on-target peptide (PE-conjugated) and off-target peptide (APC-conjugated). Cells were incubated for 30 minutes at 4C and then washed and resuspended in 120uL FACS buffer. All flow cytometry experiments were performed on the Attune NxT instrument and data was analyzed using the FlowJo™ software.

[0556] Activation assays using flow cytometry

[0557] Antigen presenting cells (APCs) (HEK293T cells presenting HLA-A*02:01, K652 cells presenting HLA-A*03:01) were resuspended into RPMI medium at 1 million cells / mL and pulsed with peptide to final concentration lOuM (ordered from Genscript and eluted in DMSO) or equivalent volume of DMSO. 100 uL of cells were added to 96-well plates. 100 uL of Jurkat-CAR cells at 1 million cells / mL were added to MHC-presenting cells. Jurkat-CAR cells and MHC-presenting cells were incubated overnight for 12 to 18 hours. After incubation, cells were centrifuged at 800g for 2 minutes to remove supernatant. Cells were stained with 50uL reagent containing anti -mouse CD69 antibody (Biolegend, 104513) conjugated with APC in FACS buffer at 1 : 100 v / v for 45 minutes in 4C, then washed and resuspended in FACS buffer. CAR-Jurkat activation was measured via flow cytometry (Attune NxT) and data was analyzed using the FlowJo™ software.

[0558] Global peptide scanning

[0559] A yeast display HLA-A1 library was generated as previously described (30) to display 9-mer peptides, with P3 and P9 as anchoring residues with limited diversity (P3 as aspartateor glutamate, P9 as tyrosine only). For other positions of the peptide library, an NNK codon was used to allow all 20 amino acids. Protein binders were expressed and purified as described with C-terminal 6XHIS tag, then biotinylated. The yeast-display HLA-A1 peptide library was selected with streptavidin-coated magnetic beads coated with biotinylated binder proteins as previously described 30).

Claims

We claim1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59, wherein the polypeptide binds to a target peptide-MHC complex noted in Table 1.

2. The polypeptide of claim 1, comprising an amino acid sequence at least 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59.

3. The polypeptide of claim 1, comprising an amino acid sequence at least 80% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59.

4. The polypeptide of claim 1, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-17, 19-40, and 56-59.

5. The polypeptide of any one of claims 1-4, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence.

6. The polypeptide any one of claims 1-4, wherein at least 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, or all identified interface residues are identical (not substituted), relative to the reference sequence7. The polypeptide of any one of claims 1-6, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all specific residues are identical (not substituted), or conservatively substituted, relative to the reference sequence.

8. The polypeptide of any one of claims 1-6, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all specific residues are identical (not substituted), relative to the reference sequence.

9. The polypeptide of any one of claims 1-8, wherein all interface residues and all specific residues are identical (not substituted), relative to the reference sequence10. The polypeptide of any one of claims 1-9, wherein substitutions relative to the reference sequence are conservative amino acid substitutions.

11. A fusion protein, comprising:(a) the polypeptide of any one of claims 1-10; and(b) one or more functional domains at the N-terminus and / or at the C-terminus of the polypeptide.

12. The fusion protein of claim 11, wherein the fusion protein comprises a chimeric antigen receptor (CAR).

13. The fusion protein of claim 12, comprising the formula X1-X2-X3-X4, wherein XI comprises the polypeptide of any one of claims 1-10;X2 comprises a hinge domain;X3 comprises a transmembrane domain;X4 comprises an intracellular domain;optionally including an amino acid linker between 1, 2, or all 3 of X1-X2; X2-X3; and X3-X4.

14. The fusion protein of claim 13, wherein one or more of the following is true:(i) the hinge domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:43;(ii) the transmembrane domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44; and / or(iii) the intracellular domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, linked in N- to C-terminal order.

15. The fusion protein of any one of clams 12-14, comprises the formula Z1-Z2-Z3, wherein:(a) Z1 comprises the amino acid sequence at least 50%, 75%, 90%, or 100% identical to the amino acid sequence of SEQ ID NO: 60;(b) Z2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the polypeptide of any embodiment or combination of embodiments herein; and(c) Z3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61.

16. The fusion protein of claim 11, wherein the fusion protein comprises a bi-specific T-cell engager of the general formula X1-X2-X3, wherein XI comprises the polypeptide of any one of claims 1-9, X2 comprises an Fc CH2 domain, and X3 comprises an Fc CH3 domain, wherein XI, X2 and X3 can be in any order in the fusion protein.

17. The fusion protein of claim 16, wherein the domain order is X1-X2-X3.

18. The fusion protein of claim 16 or 17, wherein:(a) X2 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and(b) X3 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

19. The fusion protein of any one of clams 16-18, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52.

20. The polypeptide, fusion protein, or dimer of any one of claims 1-119, wherein the polypeptide binds its target with nanomolar affinity.

21. A kit comprising:(a) the fusion protein of claim 18 or 19; and(b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53.

22. A dimer comprising:(a) the fusion protein of claim 18 or 19; and(b) a protein comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52 or 53.

23. A nucleic acid encoding the polypeptide or fusion protein of any one of claims 1-20.

24. An expression vector comprising the nucleic acid of claim 23 operatively linked to a suitable control sequence, such as a promoter.

25. A host cell comprising the polypeptide, fusion protein, nucleic acid, expression vector, and / or dimer of any preceding claim.

26. The host cell of claim 25, wherein the host cell is a T-cell.

27. The host cell of claim 16, wherein the T-cell comprises the fusion protein of any one of claims 12-15, and / or a nucleic acid and / or expression vector encoding the fusion protein of any one of claims 12-15.

28. A pharmaceutical composition, comprising:(a) the polypeptide, the fusion protein, the nucleic acid, the expression vector, the host cell, the kit, and / or the dimer of any one of claims 1-27; and(b) a pharmaceutically acceptable carrier.

29. A method for treating or limiting a disorder associated with a target pMHC in Table 1, comprising administering to a subject in need thereof an amount effective to treat or limitdevelopment of the disorder of the polypeptide, fusion protein, nucleic acid, expression vector, cell, dimer, or pharmaceutical composition of any one of claims 1-20 and 21-28.

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