System and compositions comprising mhc scaffold and methods of identifying antigens using the same

Abstract

The disclosure includes compositions and pharmaceutical compositions and kits comprising amino acid sequences that bind to antigens in vivo and in vitro. The exceptional binding through and with MHC molecules allows for screens for novel epitopes but also therapeutic applications for cell targeting. In some embodiments, the disclosure relates to methods of treating subjects with pharmaceutical compositions comprising the amino acid sequences.

Description

DOCKET NO. STFD-002-PCT PCT APPLICATION SYSTEM AND COMPOSITIONS COMPRISING MHC SCAFFOLD AND METHODS OF IDENTIFYING ANTIGENS USING THE SAME STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This disclosure was made with government support under grant number R01 AI143997 awarded by the National Institutes of Health. The government has certain rights in this disclosure. CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. provisional application No.63 / 594,297, which was filed October 30, 2023, is titled “System and Compositions Comprising MHC Scaffold and Methods of Identifying Antigens Using the Same, “ and is incorporated by reference in its entirety. SEQUENCE LISTING

[0003] The Sequence Listing filed herewith has the filename STFD-002-PCT_SL.xml, was created on October 30, 2024, has a file size of 452,320 bytes, and is incorporated herein by reference in its entirety. FIELD

[0004] The disclosure relates to compositions comprising one or a plurality of amino acid sequences or cells expressing the same, and methods of using the one or plurality of amino acid sequences for selecting and screening antigens associated with a MHC-I MR-1 or MHC II complexes. The disclosure also relates to methods of making the same amino acids and kits comprising the one or plurality of amnio acids. BACKGROUND

[0005] Adaptive immune responses employ T-cell receptors (TCRs) to recognize antigen-loaded major histocompatibility complex (MHC) molecules, allowing access to intracellular targets expressed in cancer, infectious, and autoimmune diseases. However, engineering specific TCRs and antibody-based TCR mimetics remains challenging. Production of therapeutic moleculesDOCKET NO. STFD-002-PCT PCT APPLICATION recognizing antigens on infected cells remains a problem with vaccines development and CAR-T cell manufacture. SUMMARY

[0006] The disclosure relates to a de novo platform, dubbed Targeted Recognition of Antigen- MHC Complex Reporter (TRACeR), for facile targeting of peptide antigens displayed on polymorphic HLA allotypes and metabolites on oligomorphic MR1 molecule, using a single flexible loop to confer antigen specificity. We identified a TRACeR sequence that demonstrated unprecedented specificity against peptide antigens presented by HLA-A*02:01, owing to a novel antigen recognition mode that is distinct from known immunoglobulin-based binders such as TCRs and monoclonal antibodies. Using TRACeR as a targeting component, we successfully generated bi-specific T cell engagers and chimeric antigen receptor T cells and demonstrated their potent ability to kill human cancer cell lines expressing corresponding antigens. Our platform can lead to new modalities for clinical theragnostic applications targeting both classical and nonclassical MHC antigens.

[0007] From a molecular engineering perspective, we postulate that there are alternative design strategies to target MHC-I and related antigens, beyond the established TCR docking mode. The fundamental requirements for building a binding platform de novo are: docking to an MHC in an orientation with access to the antigen, and providing a malleable surface to adapt to the conformation and surface chemistry of the peptide. Additional considerations concerning developability, stability, and specificity are also crucial. While TCRs rely primarily on the paired CDR3 loops to interact with antigens, we seek to develop a solution that would use a single, contiguous segment to interact with MHC antigens. Here, we create a de novo protein platform, which can achieve facile development of highly specific binders for antigens across different HLA allotypes and MR1 molecules. The design inspiration came from our engineering efforts on a peptide / MHC-II (pMHC-II) binding platform, which leverages a unique superantigen to achieve specific MHC-II antigen recognition. Despite MHC-I and MR1’s structural homology with MHC- II, we explored new binding modes to accommodate respectively the different peptide conformation and small molecule antigen in MHC-I and MR1. We call this general platformDOCKET NO. STFD-002-PCT PCT APPLICATION Targeted Recognition of Antigen-MHC Complex Reporter, or TRACeR, and we annotate them as TRACeR-I and TRACeR-MR1, for MHC-I and MR1 binders, respectively. TRACeR-I and -MR1 showed exquisite antigen specificity, and they also elicited potent responses against human primary tumor cells as bi-specific anti-CD3 engagers and as CARs on T cells. These results highlight the potential for TRACeRs in targeted therapeutics to combat a range of infectious, tumor-associated and autoimmune diseases.

[0008] In some embodiments, the disclosure relates to an amino acid sequence comprising a first scaffold region and an antigen binding region. In some embodiments, the amino acid sequence is from about 100 to about 120 amino acids in length. In some embodiments, the first scaffold region comprises, in an orientation from amino terminus to carboxy terminus, KEIFNMM (SEQ ID NO:1) and CX1X2X3X4X5X6X7X8C; and X1 through X8 are any amino acid.

[0009] In some embodiments, the disclosure relates to an amino acid sequence comprising a first scaffold region, a second scaffold region and an antigen binding region. In some embodiments, the amino acid sequence is from about 100 to about 120 amino acids in length. In some embodiments, the first scaffold region comprises one or a combination of CX1X2X3X4X5X6X7X8C, CX1X2X3X4X5X6X7C, CX1X2X3X4X5X6C, CX1X2X3X4X5X6X7X8X9C, or CX1X2X3X4X5X6X7X8X9X10C, wherein X1 through X10 are any amino acid. In some embodiments, the amino acid sequence GTHDK (SEQ ID NO: 7) is positioned contiguously in any five contiguous positions within X1X2X3X4X5X6X7. In some embodiments, X3,X4,X5, X6, or X7 is G. In some embodiments, X4, X5,X6, X7, or X8 is T. In some embodiments, X5, X6,X7, X8, or X9 is H. In some embodiments, X6, X7,X8, X9, or X10 is D. In some embodiments, X6, X7,X8, X9, or X10 is K. In some embodiments, X2,X3,X4, X5, or X6 is G. In some embodiments, the first scaffold comprises an amino acid sequence chosen from: CX1X2X3X4X5X6X7X8C, CX1X2X3X4X5X6X7C, CX1X2X3X4X5X6C, CX1X2X3X4X5X6X7X8X9C, or CX1X2X3X4X5X6X7X8X9X10C, wherein the amino terminal C and the carboxy terminal C are bound together by a disulfide bond forming a loop secondary structure.

[0010] In some embodiments, the disclosure relates to an amino acid sequence comprising a first scaffold region and an antigen binding region, and, optionally a second scaffold region. In someDOCKET NO. STFD-002-PCT PCT APPLICATION embodiments, the amino acid sequence is from about 100 to about 120 amino acids in length. In some embodiments, the first scaffold region and the second scaffold region comprise, collectively, at least three alpha helices. In some embodiments, the first scaffold region comprises at least about 75% sequence identity to KEIFNMMFMLLWRVFRSQRIDAN (SEQ ID NO:2). In some embodiments, the amino acid sequence is about 122 amino acids in length. In some embodiments, an antigen binding region is positioned after SEQ ID NO:3 in an orientation from amino terminus to carboxy terminus. In some embodiments, antigen binding region is positioned from about 3 to about 6 amino acids downstream from SEQ ID NO:3 in the carboxy terminal direction. In some embodiments, the antigen binding region comprises at least about 7 contiguous amino acids in length and is variable in amino acid identity. In some embodiments, the antigen binding region comprises EQWVANY (SEQ ID NO:6).

[0011] In some embodiments the disclosure provides further structural characteristics useful for these amino acid structures according to the disclosure.

[0012] In some embodiments, the disclosure relates to an amino acid sequence comprising at least five contiguous amino acids chosen from X1 through X9 and comprising at least about 80% sequence identity to GTHDK (SEQ ID NO:7) of a functional variant thereof. In some embodiments, the first scaffold region further comprises a contiguous amino acid sequence comprising at least about 75% sequence identity to NVELIKFNIRVLDWIMAEADNDLCYFI (SEQ ID NO:4) or a functional variant thereof. In some embodiments, the amino acid sequence further comprises a second scaffold region in the carboxy end of the sequence comprising an amino acid sequence comprising at least about 75% sequence identity to KEETKEVLKKFKEKVNQFYRHAFDIINKYG (SEQ ID NO:5) or a functional variant thereof. In some embodiments, the amino acid sequence comprises an amino terminal sequence of DKEIA (SEQ ID NO:8) or a functional variant thereof that comprises at least about 80% sequence identity to SEQ ID NO:8. In some embodiments, a disulfide bond is position between the cysteines of SEQ ID NO:3. In some embodiments, the amino acid sequence is organized in at least about three alpha helices across a first scaffold region and a second scaffold region, wherein the first scaffold region comprises two alpha helices and the second scaffold region comprises an alpha helix and wherein the antigen binding domain comprises a non-helical loop.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0013] In some embodiments, the disclosure relates to a composition comprising any one or plurality of amino acid sequences disclosed herein. In some embodiments, the disclosure relates to an amino acid sequence comprising any one or combination of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or functional variants thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% sequence identity. In some embodiments, the disclosure relates to an amino acid sequence comprising an amino acid comprising at least 75% sequence identity to SEQ ID NO:2; an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO:4, and an amino acid sequence at least 75% sequence identity to SEQ ID NO:5. In some embodiments, the amino acid sequence further comprises an amino acid sequence at least 75% sequence identity to SEQ ID NO:5. In some embodiments, the amino acid sequence comprises at least a first scaffold domain comprising an amino acid comprising at least 75% sequence identity to SEQ ID NO:2; and an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO:4; wherein the an amino acid comprising at least 75% sequence identity to SEQ ID NO:2 and amino acid sequence comprising at least 75% sequence identity to SEQ ID NO:4 are positioned contiguously in the amino to carboxy orientation. In some embodiments, the amino acid sequence comprises a second scaffold domain that comprises amino acid sequence comprising at least 75% sequence identity to SEQ ID NO:5; or a first scaffold domain that comprises each of: an amino acid sequence at least 75% sequence identity to SEQ ID NO:2; an amino acid sequence at least 75% sequence identity to SEQ ID NO:3; and an amino acid sequence at least 75% sequence identity to SEQ ID NO:4.

[0014] In some embodiments, the composition comprises an amino acid in a monomer conformation. In some embodiments, the composition comprises one or a plurality of amino acid sequences in a dimer confirmation. In some embodiments the dimer is a homodimer.

[0015] In some embodiments, the disclosure relates to a composition comprising an amino acid sequence comprising a first scaffold region and a second scaffold region with an antigen binding region positioned therebetween; wherein the first and second scaffold regions are from about 40 to about 80 amino acids in length. In some embodiments, the first and scaffold region comprises a first alpha helix comprising X1EEX2K , wherein X1 and X2 are independently selected from either V, K, T or I. In some embodiments, the first or the second scaffold region comprise a second alphaDOCKET NO. STFD-002-PCT PCT APPLICATION helix comprising MLLWR (SEQ ID NO: 9) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:9. In some embodiments, the first or the second scaffold region comprises a third alpha helix comprising NVELIK (SEQ ID NO: 10) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:10. In some embodiments, the first scaffold region comprises one or a combination of: (i) SVEEIKKEYEERLKRFDEFVERILKETGN (SEQ ID NO:11) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:11; (ii) KEIANMARMLLWRVERSYRIDKD (SEQ ID NO:12) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:12.; and (iii) NVELIKFNIRVIDWIMAEAENDLCYFI (SEQ ID NO:13) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:13.

[0016] In some embodiments, the disclosure relates to a composition comprising an amino acid sequence comprising a first scaffold region and a second scaffold region with an antigen binding region positioned therebetween; wherein the first scaffold region and the second scaffold region comprise at least three alpha helices; and wherein the three alpha helices are from about 20 to about 40 amino acids in length. In some embodiments, the first and scaffold region comprise a first alpha helix comprising X1EEX2K , wherein X1 and X2 are independently selected from either V, K, T or I. In some embodiments, the first and second scaffold region comprise a second alpha helix comprising MLLWR (SEQ ID NO: 9) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:9. In some embodiments, the first and second scaffold region comprise a third alpha helix comprising NVELIK (SEQ ID NO: 10) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:10. In some embodiments, the first scaffold region comprises one or a combination of: (i) SVEEIKKEYEERLKRFDEFVERILKETGN (SEQ ID NO:11) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:11; (ii) KEIANMARMLLWRVERSYRIDKD (SEQ ID NO:12) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:12.; and (iii) NVELIKFNIRVIDWIMAEAENDLCYFI (SEQ ID NO:13) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:13.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0017] In some embodiments, the antigen binding domain comprises GTHDK (SEQ ID NO: 7) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:7. In some embodiments, the antigen binding domain comprises CENPKEQWVANY (SEQ ID NO:17) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:17. In some embodiments, the antigen binding domain comprises THDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN (SEQ ID NO:18) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:18.

[0018] In some embodiments, the amino acid sequence comprises one or a combination of: (i) SEQ ID NO:11 or a functional variant thereof; (ii) SEQ ID NO:12 or a functional variant thereof; (iii) SEQ ID NO:13 or functional variant thereof; (iv) SEQ ID NO:14 or a functional variant thereof; (v) SEQ ID NO:15 or a functional variant thereof; (vi) SEQ ID NO:16 or functional variant thereof; and / or (vii) SEQ ID NO: 18 or functional variant thereof.

[0019] In some embodiments, the amino acid sequence comprises a disulfide bond positioned between the first scaffold and the antigen binding region. In some embodiments the disclosure relates to compositions comprising any one or of a plurality of amino acid sequences disclosed herein and an MHC-I peptide or variant thereof, an MHC-II peptide or variant thereof, or an MR- 1 peptide or a variant thereof. In some embodiments, the composition comprises any of the disclosed amino acid sequences associated with a second amino acid sequence that is either: (i) an MHC-I peptide or variant thereof, an MHC-II peptide or variant thereof, or an MR-1 peptide or a variant thereof; or (ii) an antigen with a known or unknown amino acid sequence. In some embodiments, the composition comprises (i) any of the disclosed amino acid sequences herein; (ii) a second amino acid that is: (i) an MHC-I peptide or variant thereof, an MHC-II peptide or variant thereof, or an MR-1 peptide or a variant thereof; and (iii) a third amino acid sequence that is an antigen with a known or unknown amino acid sequence.

[0020] In some embodiments, the amino acid sequences herein are associated with the second amino acid sequence, a third amino acid sequence or both of the second amino acid sequence and the third amino acid sequences. In some embodiments, the composition comprises any of the disclosed amino acid sequences herein associated in a dimer configuration and optionally associated with one or a combination of: (ii) a second amino acid that is an MHC-I peptide orDOCKET NO. STFD-002-PCT PCT APPLICATION variant thereof, an MHC-II peptide or variant thereof, or an MR-1 peptide or a variant thereof; and (iii) a third amino acid sequence that is an antigen with a known or unknown amino acid sequence.

[0021] In some embodiments, the disclosure relates to a system comprising any amino acid sequence according to the disclosure and an antigen. In some embodiments, the amino acid sequence is immobilized to a solid substrate. In some embodiments, the disclosure relates to a cell comprising a nucleic acid sequence encoding one or more amino acid sequence according to the disclosure. In some embodiments, the disclosure relates to a cell comprising any one or more of the amino acid sequences herein.

[0022] In some embodiments, the disclosure provides a kit comprising (i) any one or more of the disclosed amino acid sequences herein or a nucleic acid sequence encoding the same; and, optionally, (ii) a second amino acid that is an MHC-I peptide or variant thereof, an MHC-II peptide or variant thereof, or an MR-1 peptide or a variant thereof. In some embodiments, the kit further comprises (iii) a third amino acid sequence that is an antigen with a known or unknown amino acid sequence. In some embodiments, the kit comprises (i) and (ii), (i) and (iii) or (i), (ii) and (iii) each in separate containers.

[0023] In some embodiments, the disclosure relates to a method of screening a library of antigens for an epitope, antigen, or antigen binding fragment that associates with one or the amino acid sequence disclosed herein, the method comprising: (a) exposing the one or more amino acids to an antigen for a time period and in physiologically relevant conditions to allow the antigen to associate with or bind to the one more amino acid sequences to form a complex. In some embodiments, the method further comprises (b) isolating the complex; (c) performing one or more assays to identify a portion or the antigen that associates with the antigen-binding region of the one or more amino acid sequences. In some embodiments the step of performing one or more assays comprises performing one or a combination of: elution, amino acid sequencing, crystallography, artificial-intelligence (AI)- assisted in silico modeling, atomic force microscopy, surface plasmon resonance, enzyme-linked immunosorbent assays, and enzymatic protein digestion of a portion of the antigen no associated with the one or more amino acid sequences disclosed herein.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0024] In some embodiments, the disclosure relates to a method of performing X-ray crystallography of a peptide comprising: (a) exposing one or more amino acid sequences herein to an antigen for a time period and in physiologically relevant conditions to allow the antigen to associate with or bind to the one more amino acid sequences to form a complex. In some embodiments, the method further comprises (b) isolating the complex; and (c) performing x-ray crystallography on the complex.

[0025] In some embodiments, the disclosure relates to a method of identifying a protein-protein interaction, the method comprising: (a) exposing one or more amino acid sequences herein to an antigen for a time period and in physiologically relevant conditions to allow the antigen to associate with or bind to the one more amino acid sequences to form a complex. In some embodiments, the method further comprises (b) isolating the complex and / or (c) performing one or more assays to identify a portion or the antigen that associates with the antigen-binding region of the one or more amino acid sequences. In some embodiments the step of performing one or more assays comprises performing one or a combination of: elution, amino acid sequencing, crystallography, artificial-intelligence (AI)- assisted in silico modeling, atomic force microscopy, surface plasmon resonance, enzyme-linked immunosorbent assays, and enzymatic protein digestion of a portion of the antigen associated with the one or more amino acid sequences disclosed herein. In some embodiments, step (a) is performed by surface plasmon resonance or by ELISA. In some embodiments, the method further comprises a step of sequencing a portion of the antigen associated with the one or plurality of amino acid sequences after performing step (a).

[0026] In some embodiments, the disclosure relates to a method of making any one amino acid sequence herein or a plurality of amino acid sequences herein comprising expressing a nucleic acid sequence encoding the one amino acid sequence herein or the plurality of amino acid sequences herein in a cell comprising the nucleic acid sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings certain embodiments. It is understood, however,DOCKET NO. STFD-002-PCT PCT APPLICATION that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0028] FIGS.1A–1D show an overview of the TRACeR platform design scheme. FIG.1A shows a design principle: the combinatorial complexity resulted from the antigen-MHC pairing is highly challenging for conventional binding mechanisms. An antigen focused binding mode would be ideal for large scale creation of binders for disease relevant antigens. FIG. 1B: The TRACeR platforms for MHC-I and MR1 were developed based on a superantigen-inspired pMHC-II targeting platform, with extensive engineering effort to redesign the scaffold to bind MHC-I / MR1. Interface area is darker sha (FIG.1B, left panel, front helix). FIG.1C shows that the MHC binding domain of MAM can be segmented into a rigid 3-helical bundle (center) and a long flexible loop with more fluid behavior (right, top). The flexible region that we focused to embed library positions is depicted in the bottom right panel (depicted in an unwound strand with partial helical structure). FIG.1D: Schematic of our TRACeR development approach. In step 1, global docking was performed with PatchDock seeded RifDock (Appendix 1). In step 2, an iterative RosettaDock and FastDesign was performed to generate sequence diversity on the binding interface (Appendix 2). Output models were evaluated by binding energy, binding energy to antigen, shape complementarity, buried hydrogen bonds, and contact area (Appendix 3). The outputs of step 4 are shown in FIG.2.

[0029] FIGS. 2A and 2B show cross-allele specificity determination and biophysical characterization. FIG.2A shows target specificity determined with yeast surface display. Antigen- focused MHC-I and MR1 binders show high specificity with their cognate antigen and show minimum cross-reactivity with other targets. (Staining concentration: 50 nM tetramer). FIG.2B shows binding kinetics of TRACeR molecules determined by BLI. Binding kinetics and fitting quality are summarized in Table 2.

[0030] FIGS. 3A through 3D show determination of the specificity of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ with different peptides. FIG. 3A shows an alanine scan of the NY-ESO-1 antigen showing the requirement of S1, M4 and W5. Residues S1A, M4A, W5A, QBA are solvent exposed and LS1, L3A, 16A, and T7A have side chains buried in MHC-I groove. FIG.3B shows X-scan on the three key residues for TRACeR (left) and 1G4 TCR (right), MFI (mean fluorescence intensity) signalDOCKET NO. STFD-002-PCT PCT APPLICATION was normalized as a percentage of wild type binding signal (raw data shown in FIG.20A through 20F). S1V peptide synthesis failed due to the hydrophobicity. FIG.3C shows a correlation plot of the binding levels between TRACeR construct with a panel of 97 individual HLA-I allotypes upon incubation with the wild-type NY-ESO-1 or the non-binder NY-ESO-1W5A peptides. The dashed line represents a conceptual 1:1 correlation (no difference between the peptides). Raw data shown in Fig. 13A, 13B, and 13C shows alignment of MHC-I and MR1 structures. NY-ESO-1 peptide (first position shown as stick) and the 5-OP-RU antigen are as indicated by arrows in FIG. 3D. FIG.3A includes SEQ ID NO: 52.

[0031] FIGS. 4A–4E show a co-crystal structure of TRACeR-MHC revealing a unique binding mode. FIG. 4 A shows an overview of the crystal unit with a dimeric TRACeR engaging two pMHC molecules (top: top view, bottom: side view) TRACeR: Line with closed arrow head and dashed line. MHC-I heavy chain: grey, unmarked. MHC-I light chain: Line with open arrow. NY- ESO-1 peptide: labeled. FIG.4B shows antigen engagement mode comparison of 1G4 TCR (top, PDB:2BNR) and ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ. (bottom) FIG.4C shows an open view of the binding pocket of MHC-I TRACeR interface showing shape complementarity of the interface area. FIG.4D shows footprint of TRACeR binding on MHC interface. Dark grey, and labeled A158, G162, A69 and G62: pMHC interface residues that interact TRACeR. labeled A158, G162, A69 and G62: Small hydrophobic residues on MHC groove that TRACeR docks on. FIG.4E shows the antigen peptide interacting environment. Top: All TRACeR residues (light grey above N76, N76 Y112, Y72, and aromatic residues and helix to the left of W5) that interact the MHC-presented peptide (peptide including S1, W5, M4) Middle: Zoom-in view of the hydrogen bond network with S1. Bottom: Zoom-in view of the hydrogen bond with peptide backbone.

[0032] FIGS.5A through 5D show that〖TRACeR〗_(MHC-I,A02)^(NY-ESO-1)-antiCD3 scFv BiTE mediate target-specific cancer killing. FIG.5A shows a Schematic representation of the BiTE construct.〖TRACeR〗_(MHC-I,A02)^(NY-ESO-1) recognizes NY-ESO-1 antigen presented by surface MHC-I, and the anti-CD3 scFv was used for the T cell binding module. FIG.5B shows in vitro tumor cell killing assays with〖TRACeR〗_(MHC-I,A02)^(NY-ESO-1)-antiCD3 BiTEs or control BiTEs. Activated human total CD3+ cells were mixed 2:1 with tumor cell lines for 18 hours then analyzed for tumor specific killing by flow cytometry. Frequency of live tumor cellsDOCKET NO. STFD-002-PCT PCT APPLICATION were normalized to PBS treated control cells. One of two independent repeats is shown. FIGS.5C and 5D show anti-CD3 / anti-CD28 activated total T cells that were incubated with indicated tumor cell lines and treated with TRACeR BiTEs, control bispecific antibodies or PBS for 18 hours. Control bispecific antibodies were used at a concentration of 10 ng / mL. Cells were then isolated and analyzed by flow cytometry. Cells analyzed are live, CD3+CD4+ and CD3+CD8+ cells. FIG. 5C: CD69 and 4-1BB expression in T cells incubated with HBL-1 cells. FIG.5D: CD69 and 4- 1BB expression in T cells incubated with HLY-1 cells. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.

[0033] FIG. 6 depicts a schematic of one embodiment of the disclosure illustrating secondary structure and some tertiary structure of a TRACeR peptide.

[0034] FIG.7 depicts a schematic of one embodiment of the disclosure illustrating an alternative secondary structure and some tertiary structure of a TRACeR peptide.

[0035] FIG. 8 depicts a schematic alternative embodiment of the disclosure illustrating an secondary structure and some tertiary structure of a TRACeR peptide single-chain monomer. The connection scheme to create the rewired monomer. The equivalence to the crystal structure is denoted by the inverted labels on the helices (H1, H2 and H3).

[0036] FIGS.9A through 9C: FIG.9A depicts Fluorescence Activated Cell Sorting (FACS) against HLA-A*02:01 / NY-ESO-1 demonstrating MHC-1 cell specificity and activation with TRACeR-I peptide. FIG. 9A includes SEQ ID NO: 52. FIG 9B depicts cell sorting of cells activated by the TRACeR-I peptide after a screen without a negative selection step. FIG. 9C demonstrates enrichment of multiple clones from a single library that could differentiate the corresponding ligand / MR1 protein complexes. In order of appearance, SEQ ID NOS: 52–54 appear in FIG.9A– 9C.

[0037] FIG.10 depicts enrichment of multiple T cell clones stimulated by the TRACeR-MR1 sequence with antigen-specificity. FIG.10 depicts the cell sorting of cells stimulated with the TRACeR-MHCI scaffold and 1-20 deletion mutant. The cell sorting shows that the ARE and helix in the antigen-presentation area is needed for proper stimulation of T cells.

[0038] FIG.11 depicts the circular permutation scheme design of the TRACeR molecule that shifts the flexible region to an internal section of the protein, leading to a protein structure with differentDOCKET NO. STFD-002-PCT PCT APPLICATION connectivity but similar overall 3D shape as other embodiments. Bottom panels show how the circular permutated version of the TRACeR molecule with multiple antigens and paired with both MR1 and MHC-I highly activates cells to recognize antigens.

[0039] FIGS. 12A and 12B: FIG.12A depicts a protein gels after modest purification of the TRACeR proteins with Ni-NTA chromatography. Expression of TRACeR with Ecoli BL21(DE3) strain WC: whole cell, SF: soluble fraction, FT: flow through, E2, E3, E4, E5: elution fractions FIG 12B depicts circular dichroism (CD) spectra denote clear helical structures with a melting temperature of approximately 60 °C.

[0040] FIGS.13A through 13C: FIG.13A depicts an experiment using single antigen beads (SABs) displaying 97 HLA allotypes encoded with different colors. Reactivity is measured by measuring binding affinity of the beads in the presence of the TRACeR + antigen protein. The results show HLA allelic cross-reactivity landscape of〖TRACeR]-(MHC-I,A02)^(NY-ESO-1) and how that binding reactivity of beads is confirmation specific. FIG 13A shows a scatterplot of the BLOSUM62 sequence similarity to A*02:01 for HLA residues interfacing with the TRACeR versus the predicted percent rank binding affinity. The black dashed line represents a 5% rank binding affinity denoting a weak binder per NetMHCPan. FIG.13B depicts the expected crystal structure of the TRACeR embodiment bound to the antigen sequence and the HLA allele. Polymorphisms (dark spheres) present in 10 HLAs (HLA-A*02:06, HLA-A*02:07, HLA- A*02:09, HLA-A*02:11, HLA-A*02:131, HLA-A*02:17, HLA-A*02:18, HLA-A*02:240, HLA- A*02:448, HLA-A*02:642) relative to HLA-A*02:01 mapped onto the TRACeRNY-ESO-1,A02 crystal structure. FIG13C depicts a comparison o HLA-I allotypes upon incubation with the wild- type NY-ESO-1 or the non-binder NY-ESO-1W5A peptides. Binding levels of TRACeR construct with a panel of 97 individual HLA-I allotypes upon incubation with the wild-type NY-ESO-1 or the non-binder NY-ESO-1W5A peptides (Raw MFI level data of FIG.3C).

[0041] FIGS. 14A and 14B depict of the crystal structure solved for〖TRACeR〗_(MHC-I A02)^(NY-ESO-1) and HLA-A*02:01 / NY-ESO-1. FIG. 14A depicts alignment of TRACeR helical bundle (light grey and dark grey) with the helical bundle from MAM N terminal domain (grey). FIG. 14B depicts alignment of TRACeR monomeric binding mode with computational model (model is shown in shadow).DOCKET NO. STFD-002-PCT PCT APPLICATION

[0042] FIG.15 depicts functional binding affinity analysis of mutagenesis across the TRACeR molecule and shows that the hydrophobic residues at or near the ARE are responsible for binding to the MHC molecule and NY-ESO-1 antigen. Site saturate mutagenesis library of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ was stained with 10 nM pMHC monomer and top 0.5% binding population was collected as child pool. Enrichment ratio of each mutation was calculated as (sequence count percentage in child pool) / (sequence count percentage in mother pool), normalized based on the enrichment ratio of wild type. Deep sequencing was performed with Illumina 2*300 Kit at Stanford PAN facility.

[0043] FIG. 16 depicts an experiment showing comparative binding of a TRACeR embodiment binding across surface area of an antigen and MHC molecules as compared to scFvs specific for the same antigen as well as TCRs binding to the same antigen. TCR:nonamer / HLA-I structural dataset was generated using a modified version of HLA3DB1 as described previously. A selection resulted in 67 crystal structures. Complexes were analyzed using PDBePISA2 as implemented in CCP4 (v. 8.0)3 to obtain peptide / receptor and HLA / receptor interface area values. Scatter plot depicting the interface area between the immune receptor and the HLA (x-axis) or the peptide (y- axis) (light grey: TCRs, dark grey: scFvs). The corresponding interface areas of the TRACeR with HLA-A*02:01 and NY-ESO-1 peptide is shown as a triangle.

[0044] FIG.17 depicts tumor cell killing assay results after the isolating TRACeR BiTE: antiCD3 scFv and TRACeR monomer scFv embodiment from insect cells. Purification of〖TRACeR〗 _(MHC-I,A02)^(NY-ESO-1)- antiCD3 BiTEs construct is depicted by the gel image and the dimer construct shows cancer killing.

[0045] FIGS.18A through 18D depict T cells incubated with off-target tumor cell lines SUDHL4 and SUDHL5 displaying reduced, off-target activation in the presence of our TRACeR BiTE: antiCD3 scFv and TRACeR monomer embodiment in single-chain format. T cells upregulate expression of 4-1BB and CD69 when treated with dimerized TRACeR BiTEs during tumor killing assays. Anti-CD3 / anti-CD28 activated total T cells were incubated with indicated tumor cell lines and treated with TRACeR BiTEs, control bispecific antibodies or PBS for 18 hours. Control bispecific antibodies were used at a concentration of 10 ng / mL. Cells were then isolated and analyzed by flow cytometry. Cells analyzed are live, CD3+CD4+ and CD3+CD8+ cells. FIG.18ADOCKET NO. STFD-002-PCT PCT APPLICATION shows histograms depicting 4-1BB and CD69 expression in T cells incubated with SUDHL4 cells and FIG. 18B shows quantification of frequency of 4-1BB+ and CD69+ cells among CD4+ or CD8+ T cells incubated with SUDHL4. FIGS.18C and 18D show SUDHL5 cells.

[0046] FIG. 19A through 19E depict Designing dimerized TRACeR into monomer binder FIG.19A. Inspired by crystal structure, we redesigned the domain-swapped dimer into a monomer by connecting two monomers and removing one ARE site. Surface residues were redesigned with ProteinMPNN. FIG. 19B. Monomeric TRACeR purification SDS-PAGE gel and SEC curve (superdex 75). Monomeric TRACeR can be easily purified from E.coli and is highly soluble. FIG. 19C. Monomeric TRACeR is still peptide specific based on yeast surface display (staining concentration: 50 nM tetramer) FIG.19D. Titration of monomeric〖TRACeR〗_(MHC- I,A02)^(NY-ESO-1) on T2 cells pulsing NY-ESO-1 peptide or EBV peptide as negative control.

[0047] FIG.20A through 20F depict Raw fluorescence signal data for X-scan

[0048] TRACeR: TRACeR was expressed on yeast surface and staining with anti-cmyc antibody, and full scanning NY-ESO-1 library at position 1, 4, and 5 tetramer-PE.

[0049] Figure 21 depicts a crystal structure comparison of peptide interacting interface of 1G4 TCR, 3M4E5 Fab, and TRACeR molecule.

[0050] Figure 22 depicts cytometry data of FITC-stained cells using a TRACeR monomer embodiment with disulfide bridges. The purpose of the data are to estimate KD values of the monomer to cells in a screen disclosed in Example 5. DETAILED DESCRIPTION

[0051] The disclosure relates to compositions comprising an amino acid sequence that can be used to screen antigen libraries for binding MHC molecules. The disclosure relates to methods of using the composition for screening a library of antigens for detecting or calculating the magnitude of association between a MHC molecule and / or the amino acid sequence and an antigen. The disclosure relates to cells expressing the amino acid sequence as well as cells comprising the amino acid sequence. Embodiments that include cells expressing the amino acid sequence can be used for recombinant manufacture or synthesis of the amino acid sequence. Such embodiments may include methods of isolating the amino acid sequence or plurality of amino acid sequencesDOCKET NO. STFD-002-PCT PCT APPLICATION disclosed herein. In some embodiments, the disclosure relates to cell compositions comprising either one or a plurality of cells disclosed herein or a cell line comprising one or a plurality of cells disclosed herein. The disclosure also relates to a method of detecting association between one or a plurality of amino acid sequences disclosed herein and an antigen. In some embodiments, the disclosure relates to a kit comprising a composition comprising one or a plurality of amino acid sequences disclosed herein. Definitions

[0052] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), provide one skilled in the art with a general guide to many of the terms used in the present application. Additionally, the practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual “, 2nd edition (Sambrook et al., 1989); “Oligonucleotide Synthesis “ (M.J. Gait, ed., 1984); “Animal Cell Culture “ (R.I. Freshney, ed., 1987); “Methods in Enzymology “ (Academic Press, Inc.); “Handbook of Experimental Immunology “, 4th edition (D.M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene Transfer Vectors for Mammalian Cells “ (J.M. Miller & M.P. Calos, eds., 1987); “Current Protocols in Molecular Biology “ (F.M. Ausubel et al., eds., 1987); and “PCR: The Polymerase Chain Reaction “, (Mullis et al., eds., 1994).

[0053] As used in the present disclosure and claims, the singular forms “a “, “an “ and “the “ include plural forms unless the context clearly dictates otherwise.

[0054] It is understood that wherever embodiments are described herein with the language “comprising “ otherwise analogous embodiments described in terms of “consisting of “ and / or “consisting essentially of “ are also provided. It is also understood that wherever embodiments are described herein with the language “consisting essentially of “ otherwise analogous embodiments described in terms of “consisting of “ are also provided.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0055] The term “about “ as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%,, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[0056] The term “and / or “ as used in a phrase such as “A and / or B “ herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or “ as used in a phrase such as “A, B, and / or C “ is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] The term “culture vessel “ as used herein is defined as any vessel suitable for growing, culturing, cultivating, proliferating, propagating, or otherwise similarly manipulating cells. A culture vessel may also be referred to herein as a “culture insert. “ In some embodiments, the culture vessel is made out of biocompatible plastic and / or glass. In some embodiments, the plastic is a thin layer of plastic comprising one or a plurality of pores that allow diffusion of protein, nucleic acid, nutrients (such as heavy metals and hormones) antibiotics, and other cell culture medium components through the pores. In some embodiments, the culture vessel is designed to contain a hydrogel or hydrogel matrix and various culture mediums. In some embodiments, the culture vessel consists of or consists essentially of a hydrogel or hydrogel matrix. In some embodiments, the only plastic component of the culture vessel is the components of the culture vessel that make up the side walls and / or bottom of the culture vessel that separate the volume of a well or zone of cellular growth from a point exterior to the culture vessel. In some embodiments, the disclosure relates the culture vessel comprising one or a plurality of cells comprising a nucleic acid sequence encoding one or more amino acid sequences disclosed herein. In some embodiments, the cells are adherent cells.

[0058] “Crystal “ or “crystalline structure “, as used herein, refers to a solid material, whose constituent atoms, molecules, or ions are arranged in an orderly repeating pattern extending in allDOCKET NO. STFD-002-PCT PCT APPLICATION three spatial dimensions. The process of forming a crystalline structure from a fluid or from materials dissolved in the fluid is often referred to as “crystallization “ or “crystallogenesis. “ Protein crystals are almost always grown in solution. The most common approach is to lower the solubility of its component molecules gradually. Crystal growth in solution is characterized by two steps: nudeation of a microscopic crystallite (possibly having only 100 molecules), followed by growth of that crystallite, ideally to a diffraction-quality crystal.

[0059] “X-ray crystallography “, as used herein, is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and diffracts into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information, as will be known by those skilled in the art.

[0060] As used herein, the phrase “therapeutically effective amount “ means the amount of active compound or pharmaceutical agent or agent within a pharmaceutical composition that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, human physician or other clinician, such as a pathologist. The therapeutic effect is dependent upon the disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and / or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject’s response to treatment.

[0061] As used herein, the terms “treat, “ “treated, “ or “treating “ can refer to therapeutic treatment and / or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent ofDOCKET NO. STFD-002-PCT PCT APPLICATION condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0062] The term “atomic coordinates “, as used herein, refers to a set of three-dimensional co- ordinates for atoms within a molecular structure. In some embodiments, atomic coordinates are obtained using X-ray crystallography according to methods well-known to those of ordinarily skill in the art of biophysics. Briefly described, X-ray diffraction patterns can be obtained by diffracting X-rays off a crystal. The diffraction data are used to calculate an electron density map of the unit cell comprising the crystal; said maps are used to establish the positions of the atoms (i.e., the atomic co-ordinates) within the unit cell. Those skilled in the art understand that a set of structure co-ordinates determined by X-ray crystallography contains standard errors. In other embodiments, atomic co-ordinates can be obtained using other experimental biophysical structure determination methods that can include electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In yet other embodiments, atomic coordinates can be obtained using molecular modeling tools which can be based on one or more of ab initio protein folding algorithms, energy minimization, and homology-based modeling. These techniques are well known to persons of ordinary skill in the biophysical and bioinformatic arts.

[0063] The term “exposing “ as used herein refers to bringing a disclosed first protein and a cell, target receptor, second protein or other biological entity together in direct or indirect contact, in such a manner that the amino acid sequence can engage in a protein-protein interaction. This can occur directly by physical contact between the disclosed amino acid sequence acid and an MHC molecule and / or an antigen or plurality of antigens, receptor or other entity. In some embodiments, the step of exposing refers to indirect protein-protein interactions i.e., by interacting with another molecule, co-factor, factor, or protein that induces an activity of another molecule.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0064] As used herein, the term “kit “ refers to a set of components provided in the context of a system for delivering materials or diagnosing a subject with having a pancreatic cyst. Such delivery systems may include, for example, systems that allow for storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, extracellular matrix components etc. in appropriate containers) and / or supporting materials (e.g., buffers, media, cells, written instructions for performing the assay etc.) from one location to another. For example, in some embodiments, kits include one or more enclosures (e.g., boxes) containing relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit “ refers to a kit comprising two or more separate containers that each contain a subportion of total kit components. Containers may be delivered to an intended recipient together or separately. For example, a first container may contain a petri dish or polysterene plate for use in a cell culture assay, while a second container may contain cells, such as control cells. As another example, the kit may comprise a first container comprising a solid support such as a chip or slide with one or a plurality of ligands with affinities to one or a plurality of amino acid sequences disclosed herein and a second container comprising any one or plurality of reagents necessary for the detection and / or quantification of the association between the amino acid sequences of the disclosure and one or more polypeptides, such as antigen sequences from pathogens or cancer cells. The term “fragmented kit “ is intended to encompass kits containing Analyte Specific Reagents (ASR’s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contain a sub-portion of total kit components are included in the term “fragmented kit. “ In contrast, a “combined kit “ refers to a delivery system containing all components in a single container (e.g., in a single box housing each of the desired components). The term “kit “ includes both fragmented and combined kits.

[0065] As defined herein, the term “inhibition, “ “inhibit, “ “inhibiting, “ and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of expression of a certain protein within a certain pathway. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part,DOCKET NO. STFD-002-PCT PCT APPLICATION partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein in a disclosed culture, system or cell.

[0066] As used herein, the term “ligand “ or “receptor ligand “ means a molecule that specifically binds to an amino acid, either intracellularly or extracellularly. A ligand may be, without the purpose of being limitative, a protein, a (poly)peptide, a lipid, a small molecule, a protein scaffold, an antibody, an antibody fragment, a nucleic acid, a carbohydrate. A ligand may be synthetic or naturally occurring. The term “ligand “ includes a “native ligand “ which is a ligand that is an endogenous, natural ligand for a native amino acid. In most cases, a ligand is a “modulator “ that increases or decreases an intracellular response when it is in contact with, for example binds to, an amino acid that is expressed in a cell. Examples of ligands that are modulators include agonists, partial agonists, inverse agonists, and antagonists, of which a more detailed description can be found further in the specification. In some embodiments, a ligand of the disclosed amino acid sequence is an MHC molecule and / or a polypeptide that is also an antigen.

[0067] The term “conformation “ or “conformational state “ of a protein refers generally to the range of structures that a protein may adopt at any instant in time. One of skill in the art will recognize that determinants of conformation or conformational state include a protein’s primary structure as reflected in a protein’s amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, β-sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Post- translational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and cofactors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labeling,DOCKET NO. STFD-002-PCT PCT APPLICATION among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules,.W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993. A “specific conformation “ or “specific conformational state “ is any subset of the range of conformations or conformational states that a protein may adopt.

[0068] The term “hydrogel “ as used herein is defined as any water-insoluble, crosslinked, three- dimensional network of polymer chains with the voids between polymer chains filled with or capable of being filled with water. The term “hydrogel matrix “ as used herein is defined as any three-dimensional hydrogel construct, system, device, or similar structure. In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivatives or combinations thereof. In some embodiments, the hydrogel or hydrogel matrix comprises Matrigel® or vitronectin. In some embodiments, the hydrogel or hydrogel matrix can be solidified into various shapes, for example, a bifurcating shape designed to mimic a neuronal tract. In some embodiments, the hydrogel or hydrogel matrix comprises poly (ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix comprises Puramatrix. In some embodiments, the hydrogel or hydrogel matrix comprises glycidyl methacrylate-dextran (MeDex). In some embodiments, two or more hydrogels or hydrogel matrixes are used simultaneously cell culture vessel. In some embodiments, two or more hydrogels or hydrogel matrixes are used simultaneously in the same cell culture vessel but the hydrogels are separated by a wall that create independently addressable microenvironments in the tissue culture vessel such as wells. In a multiplexed tissue culture vessel it is possible for some embodiments to include any number of aforementioned wells or independently addressable location within the cell culture vessel such that a hydrogel matrix in one well or location is different or the same as the hydrogel matrix in another well or location of the cell culture vessel.

[0069] “Variant “ used herein with respect to a nucleic acid means a nucleic acid sequence comprising (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of aDOCKET NO. STFD-002-PCT PCT APPLICATION referenced nucleotide sequence or portion thereof; (iii) a nucleic acid sequence that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid sequence that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. “Variant “ with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, truncation, conservative substitution of amino acids, or addition of at least one amino acid as compared to a reference sequence, but the peptide or polypeptide retains at least one biological activity of the reference sequence upon which it is based. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No.4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. Nucleic acidDOCKET NO. STFD-002-PCT PCT APPLICATION molecules or nucleic acid sequences of the disclosure include those that encode amino acid sequences comprising one or more of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and variants or functional fragments thereof that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the coding sequences of the foregoing. The term “variant “ includes polypeptides conjugated to a non-natural chemical moieties or variants. In some embodiments, the polypeptide comprises a polymer, such as polyethylene glycol, and may be comprised of one or more additional derivitizations of cysteine, lysine, or other residues. In addition, variants of the instant disclosure may comprise a linker or polymer, wherein the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid, or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine. Polymer modification of polypeptides has been reported. U.S. Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue. WO 99 / 67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein.

[0070] The term variant also includes glycosylated variants, such as but not limited to, variants glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. In addition, splice variants are also included. The term variant also includes heterodimers, homodimers, heteromultimers, or homomultimers of any one or more polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide variants containing, for example, specific deletions or other modifications yet maintain biological activity.

[0071] In some embodiments, variants further comprise an addition, substitution or deletion that modulates biological activity of the variants. For example, the additions, substitution or deletions may modulate one or more properties or activities of the variant. For example, the additions, substitutions or deletions may modulate affinity for a receptor or binding partner, modulate (including but not limited to, increases or decreases) dimerization, stabilize receptor dimers,DOCKET NO. STFD-002-PCT PCT APPLICATION modulate the conformation or one or more biological activities of a binding partner such as an antigen or MHC molecule, modulate stability of the polypeptide, modulate cleavage by peptidases or proteases, modulate dose, modulate release or bio-availability, facilitate purification, or improve or alter a particular route of administration. Similarly, variants of the present disclosure may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification or other traits of the polypeptide.

[0072] The “percent identity “ or “percent homology “ of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. “Identical “ or “identity “ as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in aDOCKET NO. STFD-002-PCT PCT APPLICATION database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two single-stranded polynucleotides are “the complement “ of each other if their sequences can be aligned in an anti- parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5’ or the 3’ end of either sequence. A polynucleotide is “complementary “ to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0073] “Nucleic acid “ or “oligonucleotide “ or “polynucleotide “ as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementaryDOCKET NO. STFD-002-PCT PCT APPLICATION strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0074] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. In some embodiments, the nucleic acid is isolated from an organism.

[0075] The term “polypeptide “ encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full-length proteins (e.g., fully processed pro-proteins or full-length synthetic polypeptides) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).

[0076] The terms “functional fragment “ means any portion of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence that encodes any one of the nucleic acid sequences disclosed herein, and said portion encodes a polypeptide of a certain length and / or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wild-type or full-length polypeptide sequence upon which the fragment is based.

[0077] The term “salt “ refers to acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally beDOCKET NO. STFD-002-PCT PCT APPLICATION pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Acid addition salts of the compounds of the disclosure are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates can be used for example in the isolation of the compounds of the disclosure, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the disclosure are solvates and hydrates of the disclosure.

[0078] The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.

[0079] Examples of salts also include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derivedDOCKET NO. STFD-002-PCT PCT APPLICATION from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, and the like. Particularly preferred salts are sodium, lysine and arginine salts of the compounds of the disclosure. Such salts can be formed by procedures well known and described in the art. Other acids such as oxalic acid, which cannot be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the disclosure and its pharmaceutically acceptable acid addition salt. Metal salts of a chemical compound of the disclosure include alkali metal salts, such as the sodium salt of a chemical compound of the disclosure containing a carboxy group. Mixtures of isomers obtainable according to the disclosure can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallization and / or chromatographic separation, for example over silica gel or by, e.g., medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallization, or by chromatography over optically active column materials.

[0080] “Substantially complementary “ as used herein may mean that a first sequence, such as the disclosed amino acid sequences, is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0081] “Substantially identical “ as used herein may mean that, in respect to a first and a second sequence, a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0082] A “non-naturally encoded amino acid “ refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or seienocysteine. Other terms that may be used synonymouslyDOCKET NO. STFD-002-PCT PCT APPLICATION with the term “non-naturally encoded amino acid “ are “non-natural amino acid, “ “unnatural amino acid, “ “non-naturally-occurring amino acid, “ and variously hyphenated and non- hyphenated versions thereof. The term “non-naturally encoded amino acid “ also includes, but is not limited to, amino acids that occur by modification (e.g. post-translational modifications) of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrolysine and seienocysteine) but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such non-naturally-occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L -serine N acetylglucosaminyl-L- threonine and O phosphotyrosine. A very wide variety of non-naturally encoded amino acids are suitable for use in the present disclosure. Any number of non-naturally encoded amino acids can be introduced into an variant. In general, the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including but not limited to, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates. For example, a variant that includes a non-naturally encoded amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, polyethylene glycol) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting for the selective reaction of the azide and the alkyne functional groups to form a Huisgen {3+2} cycloaddition product.

[0083] The term “MHC molecule “ means an amino acid sequence from the family that code for cell surface proteins essential for the adaptive immune system. In some embodiments, the MHC molecule is an amino acid based upon a gene cluster on human chromosome 6 responsible for encoding a protein expressed on antigen-presenting cells and responsible for association and presentation of exogenous amino acids to immune effector cells. In some embodiments, the MHC molecule is chosen from a human leukocyte antigen (or “HLA “) molecule. In some embodiments, the MHC molecule is an HLA molecule that is encoded by human chromosome 6. In someDOCKET NO. STFD-002-PCT PCT APPLICATION embodiments, the MHC molecule is one amino acid set forth on Table I or a functional variant thereof. Amino Acid Sequences

[0084] The disclosure relates to amino acids comprising an antigen-binding region and one or more scaffold regions. The disclosure relates to compositions comprising the aforementioned amino acid sequence. In some embodiments, the amino acid sequence comprises from about 80 amino acids to about 400 amino acids in length. In some embodiments, the amino acid sequence comprises from about 110 amino acids to about 400 amino acids in length. In some embodiments, the amino acid sequence comprises from about 120 amino acids to about 300 amino acids in length. In some embodiments, the amino acid sequence comprises from about 110 amino acids to about 260 amino acids in length. In some embodiments, the amino acid sequence comprises from about 100 amino acids to about 300 amino acids in length. In some embodiments, the amino acid sequence comprises from about 110 amino acids to about 300 amino acids in length.

[0085] Amino acid sequences of the disclosure comprise at least a first scaffold region. In some embodiments, the scaffold region comprises at least one alpha helix structure; wherein the alpha helix provides a scaffold upon which the antigen-binding domain is exposed to potential binding partners, and in some aspects, in solution. In some embodiments, the amino acid sequence comprises at least a first and a second scaffold region, wherein the first and / or second scaffold region comprises at least one, two or three alpha helices. In some embodiments, the first scaffold region comprises about three alpha helices. In some embodiments, the second scaffold region comprises about three alpha helices. In some embodiments, the first and second scaffold region are non-contiguous in the amino acid sequence. In some embodiments, the first and second scaffold region are non-contiguous in the amino acid sequence but the first and second scaffold regions comprise from about 1 to about 3 alpha helices. In some embodiments, the first and second scaffold region are non-contiguous in the amino acid sequence but the first and second scaffold regions comprise from about 1 to about 4 alpha helices. In some embodiments, the first and second scaffold region are non-contiguous in the amino acid sequence but the first and second scaffold regions comprise from about 1 to about 5 alpha helices. In some embodiments, the first and second scaffoldDOCKET NO. STFD-002-PCT PCT APPLICATION region are non-contiguous in the amino acid sequence but the first and second scaffold regions comprise from about 1 to about 6 alpha helices.

[0086] The length of the first and / or second scaffold region may vary depending upon the embodiments. In some embodiments, the amino acid sequence comprises a first and second scaffold region, wherein the first scaffold region comprises from about 35 to about 70 amino acids in length and the second scaffold comprises from about 35 to about 70 amino acids in length. In some embodiments, the amino acid sequence comprises a first and second scaffold region, wherein the first scaffold region comprises from about 70 to about 180 amino acids in length and the second scaffold comprises from about 70 to about 180 amino acids in length. In some embodiments, the amino acid sequence comprises a first and second scaffold region, wherein the first scaffold region comprises from about 50 to about 70 amino acids in length and the second scaffold comprises from about 70 to about 180 amino acids in length.

[0087] Amino acids of the disclosure also comprise an antigen binding region. Antigen binding regions of some embodiments are configured to bind to one or plurality of antigen sequences under conditions that support association of the amino acids in the antigen binding region. In some embodiments, the antigen binding domain comprises from about 5 to about 40 amino acids in length. In some embodiments, the antigen binding domain comprises from about 5 to about 40 amino acids in length. In some embodiments, the antigen binding domain comprises from about 10 to about 35 amino acids in length. In some embodiments, the antigen binding domain comprises from about 10 to about 30 amino acids in length. In some embodiments, the antigen binding domain comprises from about 10 to about 25 amino acids in length. In some embodiments, the antigen binding domain comprises from about 15 to about 35 amino acids in length. In some embodiments, the antigen binding domain comprises from about 15 to about 30 amino acids in length. In some embodiments, the antigen binding domain comprises from about 5 to about 50 amino acids in length.

[0088] The disclosure relates to an amino acid sequence comprising antigen binding domain that is positioned toward the carboxy terminal region of the amino acid sequence relative to the first scaffold region, and relative to an orientation of amino terminus to carboxy terminus. In some embodiments, in an orientation of amino terminus to carboxy terminus, the amino acid sequenceDOCKET NO. STFD-002-PCT PCT APPLICATION comprises a first scaffold region and an antigen binding region. In some embodiments, in an orientation of amino terminus to carboxy terminus, the amino acid sequence comprises a first scaffold region, a second scaffold region and an antigen binding region. In some embodiments, the antigen binding region is positioned between the first and second scaffold region. In some embodiments, amino acids of the disclosure comprise a first scaffold region comprising three alpha helices and a second scaffold region comprising three alpha helices, wherein the first and second scaffold regions are positioned contiguously on the carboxy and amino termini of the antigen- binding domain. It should be noted that the antigen-binding domain may not physically associate with an MHC molecule or an antigen across its entire length. Rather, in some embodiments, the antigen binding region may bind across about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 or more amino acids in the antigen binding region. In some embodiment, the antigen binding region comprises SEQ ID NO:7 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:7, or a salt thereof.

[0089] In some embodiment, the antigen binding region comprises SEQ ID NO:6 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:6, or a salt thereof. In some embodiment, the antigen binding region comprises SEQ ID NO:17 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:17, or a salt thereof.

[0090] In some embodiment, the antigen binding region comprises SEQ ID NO:7 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:7, or a salt thereof; SEQ ID NO:6 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:6, or a salt thereof; and SEQ ID NO:17 or an amino acid sequence variant comprising no less than about 75% sequence identity to SEQ ID NO:17, or a salt thereof. In some embodiments, the antigen binding region comprises one or a combination of amino acids chosen from: SEQ ID NO:7 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7, or a salt thereof; SEQ ID NO:17 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:17, or a salt thereof; and / or SEQ ID NO:6 or an amino acid sequence variant comprising aboutDOCKET NO. STFD-002-PCT PCT APPLICATION 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6, or a salt thereof.

[0091] The antigen binding domain or any region of the amino acid sequence may also comprise variable amino acid positions. In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:22 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22.

[0092] SEQ ID NO:22 ZANXZNZXZVZXZNKELEDIYDESNKEETKEVLKKFZEKZZQFXEHAZDIZNKYGDZEIX ZMMXZLZ wherein Z = any amino acid wherein X = any amino acid.

[0093] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:22 or an amino acid sequence functional variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23. SEQ ID NO:23 ZANXZNZXZVZXZNKELEDIYDESNKEETKEVLKKFZEKZZQFXEHAZDIZNKYGDZEIX ZMMXZLZ wherein I wherein

[0094] In some embodiments, the antigen-binding region of the amino acid sequence comprises GTHDKCENPKXXXXXXX (SEQ ID NO:31), wherein X is any natural or non-natural amino acid, or, alternatively, the antigen-binding region of the amino acid sequence is a variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31, or a salt thereof.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0095] Amino acids sequences of the disclosure also include a first or second scaffold region. In some embodiments, the first or the second scaffold region comprises XEHAFDIINKYGDKEIXNMMXMLLXRVX (SEQ ID NO:32), wherein X is any natural or non- natural amino acid, or, alternatively, the scaffold region of the amino acid sequence is a variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32, or a salt thereof.

[0096] In some embodiments, the disclosure relates to an amino acid sequence comprising from about 70 to about 140 natural or non-natural amino acids, wherein the amino acid sequence comprises a first scaffold region, a second scaffold region and an antigen binding region, wherein the antigen-binding region comprises GTHDKCENPKXXXXXXX (SEQ ID NO:31), where X is any natural or non-natural amino acid; or, alternatively, the antigen-binding region of the amino acid sequence is a variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31, or a salt thereof; and wherein the first or second scaffold region comprises SEQ ID NO:32 or a variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32, or a salt thereof.

[0097] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:24 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24. SEQ ID NO:24 VANXQNLXNVVXTNKELEDIYDESNKEETKEVLKKFKEKVNQFXEHAFDIINKYGDKEI XNMMXMLL wherein X = any amino acid.

[0098] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:25 or an amino acid sequence variant comprising about 75%, 80%,DOCKET NO. STFD-002-PCT PCT APPLICATION 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25. SEQ ID NO:25 SVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARMLLWRVERSYRIDKDNVELIKFN IRVIDWIMAEAENDLCYFIGTHDKCENPKEQWVANXQNLXNVVXTNKELEDIYDESNKE ETKEVLKKFKEKVNQFXEHAFDIINKYGDKEIXNMMXMLL wherein X = any amino acid.

[0099] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:26 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26. SEQ ID NO:26 VANXQNLXNVVXTNKELEDIYDESNKEETKEVLKKFKEKVNQFXEHAFDIINKYGDKEI XNMMXMLLXRVXRSFRIDANNVELIKFNIRVLDWIMAEADNDLSYFISQ wherein X = any amino acid.

[0100] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:27 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27. SEQ ID NO:27 SVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARMLLWRVERSYRIDKDNVELIKFN IRVIDWIMAEAENDLCYFIGTHDKCENPKEQWVANXQNLXNVVXTNKELEDIYDESNKE ETKEVLKKFKEKVNQFXEHAFDIINKYGDKEIXNMMXMLLXRVXRSFRIDANNVELIKF NIRVLDWIMAEADNDLSYFISQ wherein X = any natural or non-natural amino acidDOCKET NO. STFD-002-PCT PCT APPLICATION

[0101] In some embodiments, the amino acid sequence comprises one or a combination of amino acids chosen from: SEQ ID NO:25 or an amino acid sequence variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25.

[0102] Some embodiments of the disclosure relate to an amino acid sequence that comprises an antigen binding region that comprises from about 10 to about 30 amino acids and at least a portion of those amino acids comprise chosen from: SEQ ID NO:18 or a variant of SEQ ID NO:18 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:18; SEQ ID NO:19 or a variant of SEQ ID NO:19 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:19; SEQ ID NO:20 or a variant of SEQ ID NO:20 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20; or SEQ ID NO:21 or a variant of SEQ ID NO:21 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21; In some embodiments, the amino acid sequence comprises a sequence chosen from: SEQ ID NO:18 or a variant of SEQ ID NO:18 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:18; SEQ ID NO:19 or a variant of SEQ ID NO:19 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:19; SEQ ID NO:20 or a variant of SEQ ID NO:20 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20; or SEQ ID NO:21 or a variant of SEQ ID NO:21 comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21; and wherein the terminal cysteines of the sequence identifier comprises a disulfide bond in at least a portion of the antigen binding region.

[0103] Some amino acid sequences of the disclosure comprise a first scaffold region, an antigen binding region and a second scaffold region in an amino terminus to carboxy terminus orientation. In some embodiments, the first scaffold region, the antigen binding region and the second scaffold region are contiguous amino acid residues. In some embodiments, amino acid sequence comprisesDOCKET NO. STFD-002-PCT PCT APPLICATION an amino terminal region and a first scaffold region, an antigen binding region and a second scaffold region in an amino terminus to carboxy terminus orientation. In some embodiments, the amino acid sequence comprises an amino terminal region from about 4 to about 14 amino acids in length. In some embodiments, the amino terminal region comprises SEQ ID NO:9 or a functional variant thereof that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.

[0104] Some amino acids of the disclosure comprise a first scaffold region and a second scaffold region, wherein the first scaffold region comprises a contiguous region of amino acid residues chosen from one or a combination of: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12; SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16, or functional variants thereof.

[0105] In some embodiments, the first scaffold region comprises amino acid residues chosen from one or a combination of: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12; or a functional variant thereof; and the second scaffold region comprises: SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16, or functional variants thereof.

[0106] In some embodiments, the first scaffold region comprises amino acid residues chosen from one or a combination of: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, or a functional variant thereof; and the second scaffold region comprises: SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12;SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16, or functional variants thereof. In some embodiments, the first scaffold region comprises amino acid residues chosen from one or a combination of: SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16, or a functional variant thereof; and the second scaffold region comprises: SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12; SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14 or functional variants thereof. In some embodiments, the first scaffold region comprises amino acid residues chosen from one or a combination of: SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16, SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12, or a functional variant thereof; and the second scaffold region comprises: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14 or functional variants thereof. In some embodiments, the first scaffold region comprises amino acid residues chosen from one or a combination of: SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:6, SEQ ID NO:12, or a functionalDOCKET NO. STFD-002-PCT PCT APPLICATION variant thereof; and the second scaffold region comprises: SEQ ID NO:4, SEQ ID NO:13; SEQ ID NO:16; SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14 or functional variants thereof. The disclosure relates to an amino acid sequence comprising SEQ ID NO:29 or a functional variant comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29. The disclosure relates to an amino acid sequence comprising SEQ ID NO:30 or a functional variant comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30. SEQ ID NO:29 DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADNDLCYFIGTHD KCENPKEQWVANYQNLNNVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFYRHAFD IINKYG SEQ ID NO:30 SVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARMLLWRVERSYRIDKDNVELIKFN IRVIDWIMAEAENDLCYFIGTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNVELI KFNIRVLDWIMAEADNDLSYFISQ SEQ ID NO:33 DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADNDLCYFIGTHD KCENPKX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNV ELIKFNIRVLDWIMAEADNDLSYFISQ wherein X1X2X3X4X5X6X7 are any natural or non-natural amino acid, and wherein X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28are consecutively QNLNNVVFTNKELEDIYDESN (SEQ ID NO: 56), or a functional variant thereof. SEQ ID NO:34 DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADNDLCYFIGTHD KCENPKX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNV ELIKFNIRVLDWIMAEADNDLSYFISQ whereinDOCKET NO. STFD-002-PCT PCT APPLICATION X1X2X3X4X5X6X7 X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28 are any natural or non-natural amino acid.

[0107] In some embodiments, the disclosure relates to an amino acid sequence comprising SEQ ID NO:33 or SEQ ID NO:34, or a functional variant thereof that comprises about at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:33 or comprises about at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:34.

[0108] Amino acid sequences of the disclosure also include amino acid sequences comprising a first scaffold region and a second scaffold region, wherein the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:16, SEQ ID NO:14, SEQ ID NO:15, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:14, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:16, SEQ ID NO:15, SEQ ID NO:14, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:15, SEQ ID NO:14, SEQ ID NO:16, or functional variants thereof. In some embodiments, the amino acid sequence further comprises an antigen-binding region comprising SEQ ID NO: 31, or a functional variant thereof. In some embodiments, the antigen-binding region is positioned between the first and second scaffold regions, oriented in amino to carboxy group orientation.

[0109] Alternatively, and in some embodiments, amino acid sequences of the disclosure also include amino acid sequences comprising a first scaffold region and a second scaffold region, wherein the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or functional variants thereof, wherein the functional variants are at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,DOCKET NO. STFD-002-PCT PCT APPLICATION 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:11 and / or SEQ ID NO:12 and / or SEQ ID NO:13. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:13, SEQ ID NO:11, SEQ ID NO:12, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:11, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:13, SEQ ID NO:12, SEQ ID NO:11, or functional variants thereof. In some embodiments, the first scaffold and second scaffold regions comprise, in an amino group to carboxy group orientation, SEQ ID NO:12, SEQ ID NO:11, SEQ ID NO:13, or functional variants thereof. In some embodiments, the amino acid sequence further comprises an antigen-binding region comprising SEQ ID NO: 31, or a functional variant. In some embodiments, the antigen-binding region is positioned between the first and second scaffold regions, oriented in amino to carboxy group orientation.

[0110] In some embodiments, the amino acid sequence comprises a first scaffold region, an antigen binding region, and a second scaffold region, in an amino terminus to carboxy terminus orientation; wherein the first scaffold region comprises one or a combination of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or functional variants thereof; wherein the second scaffold region comprises one or a combination of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or functional variants thereof; and wherein the antigen-binding region comprises one or a combination of: SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:31, or functional variants thereof. In each of the above-identified embodiments, any variants can be at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the above sequence identifiers upon which the variants are based.

[0111] All tautomeric forms and isomeric forms and mixtures, whether individual geometric isomers or stereoisomers or racemic or non-racemic mixtures, of a chemical structure or variant are intended to be encompassed by the embodiments termed “amino acids, “ unless the specificDOCKET NO. STFD-002-PCT PCT APPLICATION stereochemistry or isomeric form is specifically indicated in the variant name, chemical name or structure. All such isomeric forms of these compositions are included in the present disclosure unless expressly provided otherwise. In some embodiments, the variants of the disclosure are also represented in multiple tautomeric forms, in such instances, the disclosure includes all tautomeric forms of the variants described herein (e.g., if alkylation of a ring system results in alkylation at multiple sites, the disclosure includes all such reaction products). All such isomeric forms of such variants are included in the present disclosure unless expressly provided otherwise. All crystal forms of the variants described herein are included in the present disclosure unless expressly provided otherwise. All deuterated form of the variants described herein are included in the present disclosure. In some embodiments at least one hydrogen atom of the variant is replaced with a deuterium atom. In some embodiments at least one hydrogen atom that is involved with a hydrogen-bond is replaced with a deuterium atom. In some embodiments at least one solvent exchangeable hydrogen atom is replaced with a deuterium atom. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 1% to about 100% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 90% to about 100% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 80% to about 90% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 70% to about 80% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 60% to about 70% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 50% to about 60% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 40% to about 50% of their hydrogen replaced with deuterium atoms. In some embodiments, the compositions, pharmaceutical compositions, and variants contained therein comprise from about 30% to about 40% of their hydrogen replaced with deuterium atoms. In some embodiments, theDOCKET NO. STFD-002-PCT PCT APPLICATION compositions, pharmaceutical compositions, and variants contained therein comprise from about 20% to about 30% of their hydrogen replaced with deuterium atoms. Cells

[0112] The disclosure relates to a cell comprising a nucleic acid sequence encoding any of the amino acid sequences disclosed herein. The disclosure also relates to a cell comprising a nucleic acid sequence encoding an amino acid sequence comprising about at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:33 or SEQ ID NO:34. The disclosure also relates to a method of making or manufacturing the amino acids disclosed herein comprising culturing a cells comprising a nucleic acid sequence encoding one or a plurality of amino acids disclosed herein.

[0113] Methods of the disclosure are suitable for any kind of cultivation and any cultivation scale. For example, in some embodiments the method is used for continuous or fed-batch processes; in another embodiment the cultivation volume is from 100 ml up to 50,000 l, in another embodiment from about 100 Liters to 1about 10,000 Liters. The method as reported herein is useful for the production of amino acids disclosed herein with about 10% or less, or 8% or less, or 6% or less of the amino acids are glycosylated. The methods related to culturing a cell herein comprise a eukaryotic cell, wherein the cell in turn comprises a nucleic acid encoding an amino acid disclosed herein or a functional fragment thereof. The eukaryotic cell is in some embodiments chosen from: CHO cells, NS0 cells, BHK cells, hybridoma cells, PER.C6® cells, Sp2 / 0 cells, HEK cells, and insect cells. A person skilled in that art is familiar with medium compositions and components as well as nutrient concentrations required by different cells for optimal growth in addition will choose an appropriate medium for the cultivation of the cell (see e.g. Mather, J. P., et al. in Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Vol. 2 (1999) 777-785).

[0114] Animal or mammalian host cells suitable for harboring, expressing, and producing proteins for subsequent isolation and / or purification include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; and Kolkekar et al„ 1997, Biochemistry, 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHODOCKET NO. STFD-002-PCT PCT APPLICATION designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1 / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL- 1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al„ 1977, J. Gen. Virol., 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002, PNAS, 99: 13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP- G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. The cells may be mammalian cells selected from Hek293 cells or COS cells.

[0115] Exemplary non-mammalian cell lines include, but are not limited to, Sf9 cells, baculovirus- insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, Pages 1-7), plant cells such as tobacco cells, tomato cells, maize cells, algae cells, or yeasts such as Saccharomyces species, Schizosaccharomyces species, Hansenula species, Yarrowia species or Pichia species. In particular, the eukaryotic cells may be yeast cells from a Saccharomyces species (e.g. Saccharomyces cerevisiae), Schizosaccharomyces sp. (for example Schizosaccharomyces pombe), a Hansenula species (e.g. Hansenula polymorpha), a Yarrowia species (e.g. Yarrowia lipolytica), a Kluyveromyces species (e.g. Kluyveromyces lactis), a Pichia species (e.g. Pichia pastoris), or a Komagataella species (e.g. Komagataella pastoris). The eukaryotic cells may be Pichia cells, and most particularly e Pichia pastoris cells.

[0116] Transfection of target cells (e.g. mammalian cells) can be carried out following principles outlined by Sambrook and Russel (Molecular Cloning, A Laboratory Manual, 3rd Edition, VolumeDOCKET NO. STFD-002-PCT PCT APPLICATION 3, Chapter 16, Section 16.1-16.54). In addition, viral transduction can also be performed using reagents such as adenoviral vectors. Selection of the appropriate viral vector system, regulatory regions and host cell is common knowledge within the level of ordinary skill in the art. The resulting transfected cells are maintained in culture or frozen for later use according to standard practices.

[0117] Accordingly, another aspect of the specification relates to a method for producing a binding domain as described herein, the method comprising at least the steps of: (i) expressing in a suitable cellular expression system (as defined hereinabove) a nucleic acid as described herein, and optionally (ii) isolating and / or purifying said binding domain.

[0118] The disclosure relates to cells and, eukaryotic or prokaryotic cells that produce or are configured to produce recombinant the amino acid sequences disclosed herein.

[0119] In some embodiments, the composition comprises no less than about 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, or 75,000 cells. In some embodiments, the composition comprises no less than 75,000 cells. In some embodiments, the composition comprises no less than 65,000 cells. In some embodiments, the composition comprises no less than 60,000 cells. In some embodiments, the composition comprises no less than 100,000 cells. In some embodiments, the composition comprises no less than 125,000 cells. In some embodiments, the composition comprises no less than 150,000 cells. In some embodiments, the composition comprises no less than 175,000 cells. In some embodiments, the composition comprises no less than 200,000 cells. In some embodiments, the composition comprises no less than 225,000 cells. In some embodiments, the composition comprises no less than 250,000 cells. In some embodiments, the composition comprises no less than 12,500 cells. In some embodiments, the composition comprises from about 12,500 cells to about 250,000 cells. In some embodiments, the composition comprises from about 12,500 cells to about 100,000 cells. In some embodiments, the composition comprises from about 12,500 cells to about 75,000 cells.

[0120] Recombinant DNA methods for producing and screening for polypeptides, such as the binding molecules described herein, are known in the art (e.g. U.S. Pat. No. 4,816,567). DNA encoding the amino acids disclosed herein, the MHC molecules disclosed herein, and bindingDOCKET NO. STFD-002-PCT PCT APPLICATION molecules or fragments thereof, for example, DNA or RNA encoding a scaffold region, a antigen- bending region, or combinations thereof can be inserted into a suitable expression vector, which can then be transfected into a suitable host cell, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce an amino acid of the disclosure, to obtain the amino acid or the MHC molecule.

[0121] Suitable expression vectors are known in the art. An expression vector can contain a polynucleotide that encodes an antibody linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule or antibody-like molecules (see, e.g., U.S. Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and the variable domains or regions of the amino acid sequence may be cloned into such a vector for expression of the entire first or second scaffold regions, the entire scaffold and / or antigen-binding region, or both the first and second scaffold chains. The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can be cultured by conventional techniques to produce the binding molecule.

[0122] Mammalian cell lines suitable as hosts for expression of recombinant antibodies are known in the art and include many immortalized cell lines available from the American Type Culture Collection, including but not limit to CHO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the binding molecule. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells. Human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. Additional cell lines which may be used as hosts for expression of recombinant amino acidsDOCKET NO. STFD-002-PCT PCT APPLICATION include insect cells (e.g. Sf21 / Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia, U.S. Pat. No. 7,326,681; etc.), plants cells (U.S. 20080066200); and chicken cells (WO2008142124).

[0123] In some embodiments, cells of the disclosure comprise or express an amino acid sequence comprising one or a combination of variants or functional fragments of the biomarkers disclosed in Table 1. In such embodiments, the functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the wild-type sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% sequence identity to the amino acid sequences of Table 1.

[0124] Compositions of the disclosure include embodiments that comprise cells comprising amino acids of the disclosure or variants or functional fragments thereof that comprise no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1 through 27. Compositions of the disclosure include embodiments that comprise cells comprising amino acids of the disclosure or variants or functional fragments thereof that comprise no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 29 or SEQ ID NO:30.

[0125] The disclosure relates to TRACeR molecules disclosed herein, nucleic acid sequences encoding TRACeR molecules disclosed herein, and cells comprising TRACeR molecules disclosed herein and / or nucleic acid sequences encoding TRACeR molecules disclosed herein.

[0126] In some embodiments, the TRACeR molecule comprises or is SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441, wherein, if in any of the foregoing sequences there is an X value for the amino acid residue, X is any natural or non-natural amino acid, or functional variants thereof. In some embodiments, the functional variants comprise about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% sequence identity to SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30,DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441. In some embodiments, the disclosure relates to a TRACeR molecule disclosed here (such as any chosen from SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441 or functional variants thereof comprising at least about 75% sequence identity to SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441) fused to an anti-CD3 domain. In these embodiments, the resulting amino acid sequence is a chimeric antigen receptor, which is optionally manufactured recombinantly by a cell or exposed to a cancer cell. In such an embodiment, the anti-CD3 domain or functional variant thereof is capable of associating with CD3 on the surface of an immune cell and the TRACeR molecule or portion thereof is capable of associating with a tumor cell, thereby enhancing cytotoxic activity of the immune cell toward the cancer cell. In some embodiments, the cytotoxic immune cell is a T cell, B cell or NK cell. In some embodiments, the TRACeR molecule is an amino acid sequence (such as SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441, or functional variants thereof comprising at least about 75%, 80%, 5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441, wherein one or more of the X residues is an amino acid capable of binding to one of the antigen sequence identified in Table Y.). In some embodiments, the amino acid sequence (such as SEQ ID NO:440 or SEQ ID NO:441, or functional variants thereof comprising at least about 75%, 80%, 5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:440 or SEQ ID NO: 441, wherein one or more of the X residues is an amino acid capable of binding to one of the antigen sequence identified in Table Y in the presence of the MHC molecule partner identified in Table X or Y. Systems

[0127] The disclosure relates to a system comprising any amino acid sequences disclosed herein and one or combination of MHC molecules. In some embodiments, the system comprises any amino acid sequences disclosed herein and one or combination of antigen sequences. In some embodiments, the system comprises any amino acid sequences disclosed herein and one orDOCKET NO. STFD-002-PCT PCT APPLICATION combination of MHC molecules and one or a combination of antigen sequences. In some embodiments, the system comprises any amino acid sequences disclosed herein and one or combination of MHC molecules and one or a combination of antigen sequences, wherein one or more of the residues chosen from: 58, 59, 62, 65, 66, 69, 72, 73, 75, 76, 80, 150, 151, 154, 155, 157, 158, 159, 161, 162, 163, 166, 167, 170, associate with the MHC molecule / MHC molecule partner. In some embodiments, the system comprises any amino acid sequences (such as the disclosed TRACeR molecule or functional variants thereof) disclosed herein and one or combination of MHC molecules and one or a combination of antigen sequences, wherein the one or more of the residues of the amino acid sequence diclosed herein associate with MHC moelcule / MHC molecule partner: E58, Y59, G62, R65, K66, A69, Q72, T73, R75, V76, T80, A150, H151, E154, Q155, R157, A158, Y159, E161, G162, T163, E166, W167, R170. In some embodiments, the amino acid seqeunce of the system comprises SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441, or functional variants thereof comprising at least about 75%, 80%, 5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441, wherein one or more of the X residues is any natural or non-natural amino acid.

[0128] Non-limiting examples of MHC molecules (or, interchangeably, molecule partners) of the disclosure include the following or functional variants that are at least about 75%, 80%, 5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to examples of Table X. TABLE X: MHC Molecules Human MHC II DM-A (SEQ ID NO:35) MGHEQNQGAALLQMLPLLWLLPHSWAVPEAPTPMWPDDLQNHTFLHTVYCQDGSPSV GLSEAYDEDQLFFFDFSQNTRVPRLPEFADWAQEQGDAPAILFDKEFCEWMIQQIGPKLD GKIPVSRGFPIAEVFTLKPLEFGKPNTLVCFVSNLFPPMLTVNWHDHSVPVEGFGPTFVSA VDGLSFQAFSYLNFTPEPSDIFSCIVTHEIDRYTAIAYWVPRNALPSDLLENVLCGVAFGL GVLGIIVGIVLIIYFRKPCSGD Human MHC II DM-B (SEQ ID NO:36)DOCKET NO. STFD-002-PCT PCT APPLICATION MITFLPLLLGLSLGCTGAGGFVAHVESTCLLDDAGTPKDFTYCISFNKDLLTCWDPEENK MAPCEFGVLNSLANVLSQHLNQKDTLMQRLRNGLQNCATHTQPFWGSLTNRTRPPSVQ VAKTTPFNTREPVMLACYVWGFYPAEVTITWRKNGKLVMPHSSAHKTAQPNGDWTYQ TLSHLALTPSYGDTYTCVVEHTGAPEPILRDWTPGLSPMQTLKVSVSAVTLGLGLIIFSLG VISWRRAGHSSYTPLPGSNYSEGWHIS Human MHC II DO-A(SEQ ID NO:37) MALRAGLVLGFHTLMTLLSPQEAGATKADHMGSYGPAFYQSYGASGQFTHEFDEEQLF SVDLKKSEAVWRLPEFGDFARFDPQGGLAGIAAIKAHLDILVERSNRSRAINVPPRVTVLP KSRVELGQPNILICIVDNIFPPVINITWLRNGQTVTEGVAQTSFYSQPDHLFRKFHYLPFVP SAEDVYDCQVEHWGLDAPLLRHWELQVPIPPPDAMETLVCALGLAIGLVGFLVGTVLII MGTYVSSVPR Human MHC II DO-B (SEQ ID NO:38) MGSGWVPWVVALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKVQFVVRFIF NLEEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFT VGRKVQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGPIR NGDWTFQTVVMLEMTPELGHVYTCLVDHSSLLSPVSVEWRAQSEYSWRKMLSGIAAFL LGLIFLLVGIVIQLRAQKGYVRTQMSGNEVSRAVLLPQSC Human MHC II DP-A1 (SEQ ID NO:39) MRPEDRMFHIRAVILRALSLAFLLSLRGAGAIKADHVSTYAAFV QTHRPTGEFMFEFDEDEMFYVDLDKKETVWHLEEFGQAFSFEAQGGLANIAILNNNLN TLIQRSNHTQATNDPPEVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVT EGVAESLFLPRTDYSFHKFHYLTFVPSAEDFYDCRVEHWGLDQPLLKHWEAQEPIQMP ETTETVLCALGLVLGLVGIIVGTVLIIKSLRSGHDPRAQGTL Human MHC II DP-B1(SEQ ID NO:40) MMVLQVSAAPRTVALTALLMVLLTSVVQGRATPENYLFQGRQECYAFNGTQRFLERYIY NREEFARFDSDVGEFRAVTELGRPAAEYWNSQKDILEEKRAVPDRMCRHNYELGGPMT LQRRVQPRVNVSPSKKGPLQHHNLLVCHVTDFYPGSIQVRWFLNGQEETAGVVSTNLIR NGDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSARSKTLTGAGGFV LGLIICGVGIFMHRRSKKVQRGSA Human MHC II DQ-B1(SEQ ID NO:41) MSWKKALRIPGGLRAATVTLMLAMLSTPVAEGRDSPEDFVYQFKAMCYFTNGTERVRY VTRYIYNREEYARFDSDVEVYRAVTPLGPPDAEYWNSQKEVLERTRAELDTVCRHNYQL ELRTTLQRRVEPTVTISPSRTEALNHHNLLVCSVTDFYPAQIKVRWFRNDQEETTGVVSTPDOCKET NO. STFD-002-PCT PCT APPLICATION LIRNGDWTFQILVMLEMTPQHGDVYTCHVEHPSLQNPITVEWRAQSESAQSKMLSGIGG FVLGLIFLGLGLIIHHRSQKGLLH Human MHC II DQ-B2(SEQ ID NO:42) MSWKMALQIPGGFWAAAVTVMLVMLSTPVAEARDFPKDFLVQFKGMCYFTNGTERV RGVARYIYNREEYGRFDSDVGEFQAVTELGRSIEDWNNYKDFLEQERAAVDKVCRHNY EAELRTTLQRQVEPTVTISPSRTEALNHHNLLVCSVTDFYPAQIKVRWFRNDQEETAGV VSTSLIRNGDWTFQILVMLEITPQRGDIYTCQVEHPSLQSPITVEWRAQSESAQSKMLSGI GGFVLGLIFLGLGLIIRHRGQKGPRGPPPAGLLH Human MHC I HLA-A (SEQ ID NO: 43) MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFV RFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAG SHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWE AAHEAEQLRAYLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWAL GFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHE GLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAA SSDSAQGSDVSLTACKV Human MHC I HLA-B (SEQ ID NO: 44) MLVMAPRTVL LLLSAALALT ETWAGSHSMR YFYTSVSRPG RGEPRFISVG YVDDTQFVRFDSDAASPREE PRAPWIEQEG PEYWDRNTQI YKAQAQTDRE SLRNLRGYYN QSEAGSHTLQSMYGCDVGPD GRLLRGHDQY AYDGKDYIAL NEDLRSWTAA DTAAQITQRK WEAAREAEQRRAYLEGECVE WLRRYLENGK DKLERADPPK THVTHHPISD HEATLRCWAL GFYPAEITLTWQRDGEDQTQ DTELVETRPA GDRTFQKWAA VVVPSGEEQR YTCHVQHEGL PKPLTLRWEPSSQSTVPIVG IVAGLAVLAV VVIGAVVAAV MCRRKSSGGK GGSYSQAACS DSAQGSDVSLTA Human MHC I HLA-C (SEQ ID NO: 45) MRVMAPRALLLLLSGGLALTETWACSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFV RFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDG SHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEA ARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALG FYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHE GLQEPLTLSWEPSSQPTIPIMGIVAGLAVLVVLAVLGAVVTAMMCRRKSSGGKGGSCSQ AACSNSAQGSDESLITCKA MHC I HLA-E (SEQ ID NO: 46) MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDN DAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTL QWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEDOCKET NO. STFD-002-PCT PCT APPLICATION AEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYP AEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPE PVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSD SAQGSESHSL MHC I HLA-F (SEQ ID NO: 47) MAPRSLLLLLSGALALTDTWAGSHSLRYFSTAVSRPGRGEPRYIAVEYVDDTQFLRFDS DAAIPRMEPREPWVEQEGPQYWEWTTGYAKANAQTDRVALRNLLRRYNQSEAGSHTL QGMNGCDMGPDGRLLRGYHQHAYDGKDYISLNEDLRSWTAADTVAQITQRFYEAEEY AEEFRTYLEGECLELLRRYLENGKETLQRADPPKAHVAHHPISDHEATLRCWALGFYPA EITLTWQRDGEEQTQDTELVETRPAGDGTFQKWAAVVVPPGEEQRYTCHVQHEGLPQP LILRWEQSPQPTIPIVGIVAGLVVLGAVVTGAVVAAVMWRKKSSDRNRGSYSQAAV MHC I HLA-G( SEQ ID NO: 48) MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFV RFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEAS SHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCE AANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCW ALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQ HEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD MR1 (SEQ ID NO:49) MGELMAFLLPLIIVLMVKHSDSRTHSLRYFRLGVSDPIHGVPEFISVGYVDSHPITTYDSV TRQKEPRAPWMAENLAPDHWERYTQLLRGWQQMFKVELKRLQRHYNHSGSHTYQRM IGCELLEDGSTTGFLQYAYDGQDFLIFNKDTLSWLAVDNVAHTIKQAWEANQHELLYQ KNWLEEECIAWLKRFLEYGKDTLQRTEPPLVRVNRKETFPGVTALFCKAHGFYPPEIYM TWMKNGEEIVQEIDYGDILPSGDGTYQAWASIELDPQSSNLYSCHVEHCGVHMVLQVP QESETIPLVMKAVSGSIVLVIVLAGVGVLVWRRRPREQNGAIYLPTPDR HLA-A11:01 (SEQ ID NO:50) GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP HLA-A03:01 (SEQ ID NO: 57) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQD AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHEAEQLRAYLDGTCVEWLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELDOCKET NO. STFD-002-PCT PCT APPLICATION HLA-B07:02 (SEQ ID NO: 58) GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY WDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP HLA-A24:02 (SEQ ID NO: 59) GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY AYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQP HLA-C07:02 (SEQ ID NO: 60) CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQS AYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEP HLA-A02:01 (SEQ ID NO: 61) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP

[0129] Systems of the disclosure include embodiments that comprise: (i) any one or combination of the amino acid sequences disclosed above; and (ii) any one or combination of an MHC molecule, or functional variant thereof. MHC molecules include those sequences set forth in Table X or functional variants thereof that comprise about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to those sequences set forth in Table X. In some embodiments, the amino acid of (i) is positioned with or associated with (ii), wherein (ii) any one or combination of an MHC molecule, or functional variant thereof. MHC molecules include those sequences set forth in Table X or functional variants thereof that comprise about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to those sequences set forth in TableDOCKET NO. STFD-002-PCT PCT APPLICATION X. In some embodiments, the amino acid sequence or sequences disclosed above and positioned in association with the MHC molecule or functional variant thereof. IN some embodiments, the amino acid (i) is SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441 or functional variants thereof.

[0130] Functional variants of the disclosure include those functional variants of an MHC molecule that are from about 5 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 200 amino acids in length.

[0131] In some embodiments, functional variants of the MHC molecule are from about 15 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 20 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 25 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 30 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 35 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 40 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 45 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 50 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 55 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 60 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 65 to about 200 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 100 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 90 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 80 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 70 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 60 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 50 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 toDOCKET NO. STFD-002-PCT PCT APPLICATION about 40 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 30 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 10 to about 20 amino acids in length. In some embodiments, functional variants of the MHC molecule are from about 5 to about 10 amino acids in length. In some embodiments, the MHC molecule is a functional fragment configured to bind to one or more of the amino acid sequences disclosed in the instant specification, including one or a combination of those amino acid sequences: SEQ ID NO:1 through SEQ ID NO:34, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO, 29, SEQ ID NO:30, SEQ ID NO:356, SEQ ID NO:440 or SEQ ID NO: 441 or a functional variant thereof.

[0132] The disclosure also relates to a system comprising: (i) any one or combination of the amino acid sequences disclosed herein; and (ii) any one or combination of MHC molecule; and (iii) any one or combination of antigen sequences. The disclosure also relates to a system comprising: (i) any one or combination of the amino acid sequences disclosed herein; and (ii) any one or combination of MHC molecule; and (iii) any one or combination of antigen sequences, wherein, if the amino acid sequence (i) has an X value for a residue, the residue is any natural or non-natural amino acid and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the X residues is capable of binding the antigen sequence. In some embodiments, the antigen sequence comprises one or a functional variant of the antigen sequences of Table Y. TABLE Y: ANTIGEN SEQUENCES Loaded Peptide Name Peptide Sequence Kras G12V-10mer VVVGAVGVGK (SEQ ID NO: 62) Kras G12V-9mer VVGAVGVGK (SEQ ID NO: 63 Kras G12C-10mer VVVGACGVGK (SEQ ID NO: 64 Kras G12C-9mer VVGACGVGK (SEQ ID NO: 65 Kras G12R-10mer VVVGARGVGK (SEQ ID NO: 66 Kras G12R-9mer VVGARGVGK (SEQ ID NO: 67 Human Immunodeficiency Virus (HIV) Nef RYPLTFGWCF (SEQ ID NO: 68 PHOX2B QYNPIRTTF (SEQ ID NO: 69DOCKET NO. STFD-002-PCT PCT APPLICATION PRAME SLLQHLIGL (SEQ ID NO: 70 Additional HIV Antigen Sequences SEQ Antigen Sequence IDProtein HXB2locatioSpecies Predicted MHC Molecule PartnerNO:nGELDRWEKI 71 Gag 11-19 human B*40:02 DRWEKIRLRPG 72 Gag 14-24 human B40 KIRLRPGGK 73 Gag 18-26 human A*03:01 IRLRPGGKK 74 Gag 19-27 human B*27:05 RLRPGGKKK 75 Gag 20-28 human A*03:01 RLRPGGKKKY 76 Gag 20-29 human A*03:01 RPGGKKHYM 77 Gag 22-30 human B*07:02 RPGGKKKYKL 78 Gag 22-31 human B*51:01 GGKKKYKLK 79 Gag 24-32 human B*08:01 KYKLKHIVW 80 Gag 28-36 human A*24:02 HLVWASREL 81 Gag 33-41 human C*08:04 LVWASRELERF 82 Gag 34-44 human A*30 WASRELERF 83 Gag 36-44 human B*35:01 LETSEGCRQI 84 Gag 51-60 human B40 ELRSLYNTV 85 Gag 74-82 human B*08:01 RSLYNTVATLY 86 Gag 76-86 human A*30:02, B*58, B*63 SLYNTVATL 87 Gag 77-85 human A*02:01, A*02:02, A*02:05 SLYNTVATLY 88 Gag 77-86 human A*02:01 LYNTVATL 89 Gag 78-85 human C*14 LYNTVATLY 90 Gag 78-86 human A*29:02, B*44:03 TLYCVHQK 91 Gag 84-91 human A*11:01 IEIKDTKEAL 92 Gag 92-101 human B*40:01 NSSKVSQNY 93 Gag 124-132 human B*35:01 VQNLQGQMV 94 Gag 135-143 human B13 GQMVHQAI 95 Gag 140-147 human B*13:02 HQAISPRTL 96 Gag 144-152 human B*15:10 QAISPRTLNAW 97 Gag 145-155 human A*25:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2locSpecies Predicted MHC Molecule PartnerNO:ationISPRTLNAW 98 Gag 147-155 human B*57:01, B*63 SPRTLNAWV 99 Gag 148-156 human B*07:02 VKVIEEKAF 100 Gag 156-164 human B*15:03 EEKAFSPEV 101 Gag 160-168 human B*44:15 KAFSPEVI 102 Gag 162-169 human B*57:03 KAFSPEVIPMF 103 Gag 162-172 human B*57:01, B*57:03, B*63 FSPEVIPMF 104 Gag 164-172 human B57 EVIPMFSAL 105 Gag 167-175 human A*26:01, A*26:02, A*26:03 VIPMFSAL 106 Gag 168-175 human C*01:02 SEGATPQDL 107 Gag 176-184 human B*40:01TPQDLNTMLB*07:02, B*39:10, B*42:01, 108Gag 180-188 humanB*81:01, C*08:02 TPQDLNMML 109 Gag 180-188 human B*53 GHQAAMQML 110 Gag 193-201 human B*15:10, B*39:01 KETINEEAA 111 Gag 202-210 human B*40:02 ETINEEAAEW 112 Gag 203-212 human A*25:01 AEWDRVHPV 113 Gag 210-218 human B*40:02 HPVHAGPIA 114 Gag 216-224 human B*35:01, B7 GQMREPRGSDI 115 Gag 226-236 human B13 TSTLQEQIGW 116 Gag 240-249 human B*57:01, B*58:01 NPPIPVGDIY 117 Gag 253-262 human B*35:01 PPIPVGDIY 118 Gag 254-262 human B*35:01 EIYKRWII 119 Gag 260-267 human B*08:01 RRWIQLGLQK 120 Gag 263-272 human B*27:03 KRWIILGLNK 121 Gag 263-272 human B*27:05 GLNKIVRMY 122 Gag 269-277 human B*15:01, B*62 VRMYSPVSI 123 Gag 274-282 human C*18 RMYSPTSI 124 Gag 275-282 human B*52:01 YSPVSILDI 125 Gag 277-285 human C*01:02 FRDYVDRFF 126 Gag 293-301 human C*18 FRDYVDRFYK 127 Gag 293-302 human B*18:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2lSpecies Predicted MHC Molecule PartnerNO:ocationRDYVDRFFKTL 128 Gag 294-304 human A*24:02 RDYVDRFYKTL 129 Gag 294-304 human B*44:02 YVDRFYKTL 130 Gag 296-304 human A*02:07 YVDRFFKTL 131 Gag 296-304 human B*15:03, C*03:03, C*03:04 DRFYKTLRA 132 Gag 298-306 human B*14:02 AEQASQDVKNW 133 Gag 306-316 human B*44:02 AEQASQEVKNWM 134 Gag 306-317 human Cw5 QASQEVKNW 135 Gag 308-316 human B*53:01, B*57:01, B*58:01 VKNWMTETL 136 Gag 313-321 human B*48:01 DCKTILKAL 137 Gag 329-337 human B*08:01 ACQGVGGPGHK 138 Gag 349-359 human A*11:01 GPGHKARVL 139 Gag 355-363 human B*07:02 AEAMSQVTNS 140 Gag 364-373 human B*45:01 CRAPRKKGC 141 Gag 405-413 human B14 APRKKGCWK 142 Gag 407-415 human B*07:02 TERQANFL 143 Gag 427-434 human B*40:02 RQANFLGKI 144 Gag 429-437 human B*48:01, B13 FLGKIWPSYK 145 Gag 433-442 human A*02:01 KELYPLTSL 146 Gag 481-489 human B*40:01 NSPTRREL 147 Pol 24-31 human C*01:02 ITLWQRPLV 148 Pol 59-67 human A*68:02, A*74:01 DTVLEEWNL 149 Pol 86-94 human A*68:02 EEMNLPGRW 150 Pol 90-98 human B44 RQYDQILIEI 151 Pol 113-122 human B13 GKKAIGTVL 152 Pol 124-132 human B*15:03 KAIGTVLV 153 Pol 126-133 human B57 LVGPTPVNI 154 Pol 132-140 human A*02:01 TPVNIIGRNML 155 Pol 136-146 human B81 FPISPIETV 156 Pol 155-163 human B*54:01 IETVPVKL 157 Pol 160-167 human B*40:01 GPKVKQWPL 158 Pol 173-181 human B*08:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2lSpecies Predicted MHC Molecule PartnerNO:ocationALVEICTEM 159 Pol 188-196 human A*02:01 ALVEICTEMEK 160 Pol 188-198 human A*03:01 KLVDFRELNK 161 Pol 228-237 human A*03:01 GIPHPAGLK 162 Pol 248-256 human A*03:01 TVLDVGDAY 163 Pol 262-270 human B*35:01 SVPLDEGFRK 164 Pol 272-281 human A*11:01 VPLDEDFRKY 165 Pol 273-282 human B*35:01 YTAFTIPSV 166 Pol 282-290 human A2 YTAFTIPSI 167 Pol 282-290 human A*02:06 TAFTIPSI 168 Pol 283-290 human B*51:01 NETPGIRYQY 169 Pol 292-301 human B18 IRYQYNVL 170 Pol 297-304 human B*14:01 IRYQYNVLP 171 Pol 297-305 human B*73 LPQGWKGSPA 172 Pol 304-313 human B*54:01 SPAIFQSSM 173 Pol 311-319 human B7 SPAIFQSSMTK 174 Pol 311-321 human A*11:01, B*07:02 AIFQSSMTK 175 Pol 313-321 human A*03:01, A*11:01 KQNPDIVIY 176 Pol 328-336 human A*30:02, C*12:02 NPEIVIYQY 177 Pol 330-338 human B18 HPDIVIYQY 178 Pol 330-338 human B*35:01 EIVIYQYMD 179 Pol 332-340 human B*18:01 VIYQYMDDL 180 Pol 334-342 human A*02:01 VIYQYMDDLYV 181 Pol 334-344 human A*02:01 IEELRQHLL 182 Pol 357-365 human B*40:01 IVLPEKDSW 183 Pol 399-407 human B*57:01 LVGKLNWASQIY 184 Pol 415-426 human B*15:01 KLNWASQIY 185 Pol 418-426 human A*30:02 QIYPGIKVR 186 Pol 424-432 human A*03:01 YPGIKVRQL 187 Pol 426-434 human B*42:01 IPLTEEAEL 188 Pol 448-456 human B*35:01, B*51:01 ILKEPVHGV 189 Pol 464-472 human A*02:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2locationSpecies Predicted MHC Molecule PartnerNO: ILKEPVHGVY 190 Pol 464-473 human B*15:01, C*12:02 GQGQWTYQI 191 Pol 488-496 human B13 IYQEPFKNLK 192 Pol 496-505 human A*11:01 RMRGAHTNDV 193 Pol 511-520 human A*30:02 RMRGAHTNDVK 194 Pol 511-521 human A*03:01 IAMESIVIW 195 Pol 530-538 human B*58:01 PIQKETWETW 196 Pol 547-556 human A*32:01 GAETFYVDGA 197 Pol 591-600 human A*68:02 ETFYVDGA 198 Pol 593-600 human A*68:02 ETFYVDGAANR 199 Pol 593-603 human A66 ETKLGKAGY 200 Pol 604-612 human A*26:01 IVTDSQYAL 201 Pol 650-658 human C*08:02 VTDSQYAL 202 Pol 651-658 human C*08 VTDSQYALGI 203 Pol 651-660 human B*15:03 QIIEQLIKK 204 Pol 675-683 human A*11:01 KLVSQGIRKV 205 Pol 705-714 human A*02:01 LFLDGIDKA 206 Pol 715-723 human B81 LPPIVAKEI 207 Pol 743-751 human B*42:01 THLEGKIIL 208 Pol 781-789 human B*15:10 HVASGYIEA 209 Pol 793-801 human B*54:01 IEAEVIPAET 210 Pol 799-808 human B*40:02 HTDNGSNF 211 Pol 829-836 human C*05 HTDNGSNFT 212 Pol 829-837 human C*05 STTVKAACWW 213 Pol 838-847 human B57 IQQEFGIPY 214 Pol 850-858 human B*15:03 VRDQAEHL 215 Pol 880-887 human C*18 KTAVQMAVF 216 Pol 888-896 human B*57:01 AVFIHNFKRK 217 Pol 894-903 human A*03:01, A*11:01 FKRKGGIGGY 218 Pol 900-909 human B*15:03 KRKGGIGGY 219 Pol 901-909 human B*27:05 GERIVDII 220 Pol 912-919 human B*40:02DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2locaSpecies Predicted MHC Molecule PartnerNO:tionIIATDIQTK 221 Pol 918-926 human A*11 LQKQITKI 222 Pol 928-935 human B*52:01 KIQNFRVYY 223 Pol 934-942 human A*30:02 YRDSRDPLW 224 Pol 942-950 human B*38 VVPRRKAKII 225 Pol 974-983 human B*08:01 VPRRKAKII 226 Pol 975-983 human B42 RKAKIIRDY 227 Pol 978-986 human B*15:03 RKAKIIRDYGK 228 Pol 978-988 human B*08:01 KIIRDYGK 229 Pol 981-988 human B*08:01 IIKDYGKQM 230 Pol 982-990 human B*42:01 RIRTWKSLVK 231 Vif 17-26 human A*03:01 IRTWKSLVK 232 Vif 18-26 human A*30:01 IRTWKSLVKH 233 Vif 18-27 human B*27:05 HMYISKKAK 234 Vif 28-36 human A*03:01 ISKKAKGWF 235 Vif 31-39 human B*57:01 HPRVSSEVHI 236 Vif 48-57 human B*07:02 IPLGDAKLII 237 Vif 57-66 human B51 WHLGHGVSI 238 Vif 79-87 human B*15:10 WHLGQGVSI 239 Vif 79-87 human B*38:01 LGHGVSIEW 240 Vif 81-89 human B*57:03 IEWRLRRY 241 Vif 87-94 human B*18:01 LADQLIHLHY 242 Vif 102-111 human B*18:01 KTKPPLPSVKK 243 Vif 158-168 human A*03:01 EAVRHFPRI 244 Vpr 29-37 human B51 AVRHFPRIW 245 Vpr 30-38 human B*57:01 VRHFPRIWL 246 Vpr 31-39 human B27 FPRIWLHGL 247 Vpr 34-42 human B*07:02, B*81:01 ETYGDTWTGV 248 Vpr 48-57 human A*68:02 DTWAGVEAIIR 249 Vpr 52-62 human A*68:01 AIIRILQQL 250 Vpr 59-67 human A*02:01 CCFHCQVC 251 Tat 30-37 human C*12DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2locationSpecies Predicted MHC Molecule PartnerNO: FQTKGLGISY 252 Tat 38-47 human B*15:03 ITKGLGISYGR 253 Tat 39-49 human A*68:01 EELLKTVRL 254 Rev 10-18 human B*44:02, B*44:03 KAVRLIKFLY 255 Rev 14-23 human B*57:01, B*58:01, B*63 QAVRIIKILY 256 Rev 14-23 human B*57:03 ERILSTYLGR 257 Rev 57-66 human A*03:01 RPAEPVPLQL 258 Rev 66-75 human B7 SAEPVPLQL 259 Rev 67-75 human C*05:01 YRLGVGALI 260 Vpu 5-13 human C*18 EYRKILRQR 261 Vpu 29-37 human A*33:03 RVKEKYQHL 262 Env 2-10 human B*08:01 AENLWVTVY 263 Env 31-39 human B*18:01, B44 AENLWVTVYY 264 Env 31-40 human B*44:02 TVYYGVPVWK 265 Env 37-46 human A*03:01 VPVWKEATTT 266 Env 42-51 human B*55:01 VPVWKEATTTL 267 Env 42-52 human B*35:01 LFCASDAKAY 268 Env 52-61 human A*24:02 KAYETEVHNVW 269 Env 59-69 human B*58 YETEVHNVW 270 Env 61-69 human B*18:01 DPNPQEVVL 271 Env 78-86 human B*35:01 MHEDIISLW 272 Env 104-112 human B*38:01 SVITQACPK 273 Env 199-207 human A*11:01 SFEPIPIHY 274 Env 209-217 human A*29:02 CAPAGFAIL 275 Env 218-226 human C*01 RPNNNTRKSI 276 Env 298-307 human B*07:02 HIGPGRAFY 277 Env 310-318 human A*30:02 RGPGRAFVTI 278 Env 311-320 human A*02:01 EIIGDIRQAY 279 Env 321-330 human A*25:01 SFNCGGEFF 280 Env 375-383 human B*15:16, C*04:01 LPCRIKQII 281 Env 416-424 human B*51:01 RIKQIINMW 282 Env 419-427 human A*32:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2Species Predicted MHC Molecule PartnerNO:locationRAIEAQQHL 283 Env 557-565 human C*03:04, C*15 RAIEAQQHM 284 Env 557-565 human C*08 QTRVLAIERYL 285 Env 577-587 human B*58:02 ERYLKDQQL 286 Env 584-592 human B*14:02 RYLKDQQLL 287 Env 585-593 human A*24:02, A23 YLKDQQLL 288 Env 586-593 human B*08:01 TAVPWNASW 289 Env 606-614 human B*35:01 VFAVLSIVNR 290 Env 698-707 human A*33:03 IVNRNRQGY 291 Env 704-712 human A*30:02 RLRDLLLIVTR 292 Env 770-780 human A*03:01, A*31:01 IVTRIVELL 293 Env 777-785 human A*68:02 GRRGWEALKY 294 Env 786-795 human B*27:05 RRGWEVLKY 295 Env 787-795 human A*01:01 KYCWNLLQY 296 Env 794-802 human A*30:02 QELKNSAVSL 297 Env 805-814 human B*40:01 SLLNATDIAV 298 Env 813-822 human A*02:01 LLNATDIAV 299 Env 814-822 human A*02:01 EVAQRAYR 300 Env 831-838 human A*33:03 IPRRIRQGL 301 Env 843-851 human B*07:02 RIRQGLERA 302 Env 846-854 human A*02:05 RQGLERALL 303 Env 848-856 human B8 WPTVRERM 304 Nef 13-20 human B*08:01 RMRRAEPAA 305 Nef 19-27 human B62 LEKHGAITS 306 Nef 37-45 human B*40:01, B50 FPVTPQVPL 307 Nef 68-76 human B*07:02 FPVTPQVPLR 308 Nef 68-77 human B*07:02 TPQVPLRPM 309 Nef 71-79 human B*07:02 RPQVPLRPM 310 Nef 71-79 human B*42:01, B*42:02 RPQVPLRPMTY 311 Nef 71-81 human B35 QVPLRPMTYK 312 Nef 73-82 human A*03:01, A*11:01 VPLRPMTY 313 Nef 74-81 human B*35:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2locaSpecies Predicted MHC Molecule PartnerNO:tionPLRPMTYK 314 Nef 75-82 human A*11:01 LRPMTYKAA 315 Nef 76-84 human B*27:03 RPMTYKAAL 316 Nef 77-85 human B*07:02 KAAFDLSFF 317 Nef 82-90 human B*57:03, B*58:01 KAAVDLSHFL 318 Nef 82-91 human Cw8 GAFDLSFFL 319 Nef 83-91 human A*02:05 AAVDLSHFL 320 Nef 83-91 human B*58, B14, C*08:02 AAFDLSFFL 321 Nef 83-91 human B*57:03 AALDLSHFL 322 Nef 83-91 human Cw3 AVDLSHFLK 323 Nef 84-92 human A*03:01, A*11:01 FLKEKGGL 324 Nef 90-97 human B*08:01 KEKGGLEGL 325 Nef 92-100 human B*40:01, B*40:02 KEKGGLEGLIY 326 Nef 92-102 human B*44:03 RRQDILDLWI 327 Nef 105-114 human B*27:05 RRQDILDLWVY 328 Nef 105-115 human B18 KRQEILDLWVY 329 Nef 105-115 human C*07 RQDILDLWI 330 Nef 106-114 human B13 RQDILDLWV 331 Nef 106-114 human B*13:02 QEILDLWVY 332 Nef 107-115 human B*44:02, B*44:03 HTQGYFPDW 333 Nef 116-124 human B*57:03, B*58:01, B57 TQGYFPDWQNY 334 Nef 117-127 human B*15:01 GYFPDWQNY 335 Nef 119-127 human B*58, C*02 YFPDWQNYT 336 Nef 120-128 human A29, B*35:01, C*06, Cw6 FPDWQNYTP 337 Nef 121-129 human B*54:01 NYTPGPGIRY 338 Nef 126-135 human A*24:02 YTPGPGIRY 339 Nef 127-135 human B*63, B57 TPGPGVRYPL 340 Nef 128-137 human B*07:02, B*42:01, B*42:02 TRYPLTFGW 341 Nef 133-141 human A33 RYPLTFGW 342 Nef 134-141 human A*24:02 RYPLTFGWCY 343 Nef 134-143 human A*24:02, C*07 YPLTFGWCY 344 Nef 135-143 human B*18:01, B*53:01DOCKET NO. STFD-002-PCT PCT APPLICATION SEQ Antigen Sequence IDProtein HXB2lSpecies Predicted MHC Molecule PartnerNO:ocationYPLTFGWCF 345 Nef 135-143 human B53 PLTFGWCYKL 346 Nef 136-145 human A*02:01 LTFGWCFKL 347 Nef 137-145 human B*63, B57 VLEWRFDSRL 348 Nef 180-189 human A*02:01 WRFDSRLAF 349 Nef 183-191 human B*15:03 SRLAFHHMA 350 Nef 187-195 human B8

[0133] Some embodiments of the disclosure relate to a system comprising one or a combination the TRACeR amino acid sequences of disclosed herein; and an antigen sequence or library of antigens sequences that comprise any one or combination of those antigen sequences identified in Table Y, or a functional variant thereof that comprises about 85% sequence identity to one of the antigen sequences identified in Table Y. In some embodiments, the system further comprises an MHC molecule. And, in some embodiments, the MHC molecule is the MHC molecule identified in Table Y which corresponds to the antigen sequence identified in Table Y. In some embodiments, the TRACeR amino acid sequence is chosen from one or a combination of two or more of: SEQ ID NO:1 through SEQ ID NO:20, SEQ ID NO:367, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441 or functional variants thereof comprising at least about 75% sequence identity to SEQ ID NO:1 through SEQ ID NO:20, SEQ ID NO:367, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441. In some embodiments, the TRACeR amino acid sequence is chosen from one or a combination of two or more of: sequence from Table 1, SEQ ID NO:1 through SEQ ID NO:20, SEQ ID NO:367, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441 or functional variants thereof comprising at least about 75% sequence identity to thos amino acid sequence of Table 1, SEQ ID NO:1 through SEQ ID NO:20, SEQ ID NO:367, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441.

[0134] The present disclosure also relates to a system comprising: (i) a cell culture vessel optionally comprising a hydrogel; and (ii) one or a plurality of cells either in suspension or as a component of an adherent culture comprising a nucleic acid sequence encoding one or a combination of the amino acid sequences of disclosed herein, such as the TRACeR molecules. InDOCKET NO. STFD-002-PCT PCT APPLICATION some embodiments, the system further comprises a nucleic acid sequence encoding an antigen sequence or a functional variant thereof. In some embodiments, the system further comprises a nucleic acid sequence or a plurality of nucleic acid sequence encoding one or a plurality of MHC molecules. In some embodiments, the system comprises one or a plurality of cells, such an immune cell, comprising the amino acid sequences or the nucleic acid sequences disclosed herein.

[0135] The disclosure also relates to a cell-free system in which peptides disclosed herein are mixed in a single container or separated in individual containers. Peptides or proteins may be alternatively made by solid state chemistry. In some embodiments, the peptide or proteins may be present in a composition comprising buffer, such as phosphate buffered saline with or without organic solvent.

[0136] In some embodiments, the disclosure relates to a system comprising a cell culture unit comprising one or a plurality of cell reactor surfaces housed in at least a first compartment, the one or plurality of cell reactor surfaces in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet; a gas transfer module in operable connection to the one or plurality of cell reactor surfaces; and a first gas inlet in operable connection to the gas transfer module.

[0137] In some embodiments, the system comprises one or plurality of cell reactor surfaces that have a surface area from about 0.2 meters squared to about 100 meters squared. In some embodiments, the one or plurality of cell reactor surfaces are configured in a cylindrical form with a hollow volume fixed within a cylindrical first compartment; wherein the first media line and the second media line are positioned on opposite faces of the cylinder. In some embodiments of the above aspects and embodiments, the first media line is attached to a first sealable aperture configured for sterile attachment of a cell culture media source. In some embodiments, the first gas inlet is attached to a second sealable aperture configured for sterile attachment of a gas source. In some embodiments, the system further comprises an apheresis unit in fluid communication with the cell culture unit. In some embodiments of the above aspects and embodiments, the system further comprises a harvesting compartment in fluid communication with the cell culture unit. In some embodiments, the system further comprises a pump and a fluid regulator in operable contactDOCKET NO. STFD-002-PCT PCT APPLICATION with the first media line, wherein the pump is capable of generating pressure in the first media line and wherein the fluid regulator is capable of regulating the speed of fluid from the pump through the first compartment and into the second media line. In some embodiments, the gas module comprises a gas pump and a gas regulator connected to the first compartment by a first gas line; wherein the first compartment comprises at least one gas outlet; wherein the gas pump is configured to generate air pressure from the pump to the first compartment through the first gas line, wherein the at least one gas outlet is a vent or in configured for sterile connection to a vent; and wherein the gas regulator is configured to regulate the speed of gas from the pump through the first compartment. In some embodiments, the system further comprises one or a plurality of cells disclosed herein and a cell expressing a MHC molecule or a TCR or a BCR.

[0138] In some embodiments, a system comprising a cell culture unit is utilized to culture the amino acids of the disclosure and expand a T-cell population described herein. In some embodiments, the composition comprises no less than about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 45,000, about 50,000, or about 60,000 cells. In some embodiments, the composition comprises from about 25,000 to about 100,000 cells. In some embodiments, the composition comprises from about 35,000 to about 100,000 cells. In some embodiments, the composition comprises from about 45,000 to about 100,000 cells. In some embodiments, the composition comprises from about 55,000 to about 100,000 cells. In some embodiments, the composition comprises from about 75,000 to about 100,000 cells.

[0139] In certain embodiments, composition is comprises from about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells,DOCKET NO. STFD-002-PCT PCT APPLICATION about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0140] In some embodiments, the cells or amino acids of the disclosure can be stored at a temperature between about −80° C. and about 16° C. (e.g., about −80° C. and about 12° C., −80° C. and about 10° C., about −80° C. and about 8° C., about −80° C. and about 6° C., about −80° C. and about 4° C., about −80° C. and about 2° C., about −80° C. and about 0° C., about −80° C. and about −4° C., about −80° C. and about −10° C., about −80° C. and about −16° C., about −80° C. and about −20° C., about −80° C. and about −25° C., about −80° C. and about −30° C., about −80° C. and about −35° C., about −80° C. and about −40° C., about −80° C. and about −45° C., about −80° C. and about −50° C., about −80° C. and about −55° C., about −80° C. and about −60° C., about −80° C. and about −65° C., about −80° C. and about −70° C., about −60° C. and about 16° C., about −60° C. and about 12° C., about −60° C. and about 10° C., about −60° C. and about 8° C., about −60° C. and about 6° C., about −60° C. and about 4° C., about −60° C. and about 2° C., about −60° C. and about 0° C., about −60° C. and about −4° C., about −60° C. and about −10° C., about −60° C. and about −10° C., about −60° C. and about −16° C., about −60° C. and about −20° C., about −60° C. and about −25° C., about −60° C. and about −30° C., about −60° C. and about −35° C., about −60° C. and about −40° C., about −60° C. and about −50° C., about −50° C. and about 16° C., about −50° C. and about 12° C., about −50° C. and about 10° C., about −50° C. and about 8° C., about −50° C. and about 6° C., about −50° C. and about 4° C., about −50° C. and about 2° C., about −50° C. and about 0° C., about −50° C. and about −4° C., about −50° C. and about −10° C., about −50° C. and about −16° C., about −50° C. and about −20° C., about −50° C. and about −30° C., about −50° C. and about −40° C., about −20° C. and about 16° C., about −20° C. and about 12° C., about −20° C. and about 10° C., about −20° C. and about 8° C., about −20° C. and about 6° C., about −20° C. and about 4° C., about −20° C. and about 2° C., about −20° C. and about 0° C., about −20° C. and about −4° C., about −20° C. and about −10° C., about −20° C. and about −15° C., about −10° C. and about 16° C., about −10° C. and about 12° C., about −10° C. and about 10° C., about −10° C. and about 8° C., about −10° C. and about 6° C., about −10° C. and about 4° C., about −10° C. and about 2° C., about −10° C. and about 0° C., about −10° C. andDOCKET NO. STFD-002-PCT PCT APPLICATION about −4° C., about −10° C. and about −6° C., about −4° C. and about 16° C., about −4° C. and about 10° C., about −4° C. and about 6° C., about −4° C. and about 4° C., about −4° C. and about 2° C., about −4° C. and about 0° C., about −2° C. and about 16° C., about −2° C. and about 12° C., about −2° C. and about 10° C., about −2° C. and about 6° C., about −2° C. and about 4° C., about −2° C. and about 2° C., about −2° C. and about 0° C., about 0° C. and about 16° C., about 0° C. and about 14° C., about 0° C. and about 12° C., about 0° C. and about 10° C., about 0° C. and about 8° C., about 0° C. and about 6° C., about 0° C. and about 4° C., about 2° C. and about 16° C., about 2° C. and about 12° C., about 2° C. and about 10° C., about 2° C. and about 8° C., about 2° C. and about 6° C., about 2° C. and about 4° C., about 4° C. and about 16° C., about 4° C. and about 12° C., about 4° C. and about 10° C., about 4° C. and about 8° C., about 4° C. and about 6° C., about 6° C. and about 16° C., about 6° C. and about 12° C., about 6° C. and about 10° C., about 6° C. and about 8° C., about 8° C. and about 16° C., about 8° C. and about 12° C., about 8° C. and about 10° C., about 10° C. and about 16° C., about 10° C. and about 12° C., or about 12° C. and about 16° C.) for about 1 day to about 7 days (e.g., about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 7 days, about 5 days to about 6 days, or about 6 days to about 7 days). Kits

[0141] The disclosure relates to kits comprising any one or plurality of amino acid sequences disclosed herein. Some embodiments, include one or plurality of amino acid sequences disclosed herein, one or a plurality of the MHC molecules disclosed herein, and / or one or a plurality fo the antigen sequence disclosed herein.

[0142] Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrates, and / or may be supplied as an electronic -readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readable memory storageDOCKET NO. STFD-002-PCT PCT APPLICATION device. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit or supplied as electronic mail.

[0143] In another embodiment, a packaged kit is provided that contains the amino acid to be utilized, optionally sealed, for housing the amino acid sequence during storage and prior to use, and instructions for carrying out in vitro methods disclosed herein in a manner effective to expose the amino acid sequences of the disclosure to antigen sequence or antigen libraries of known or unknown antigens. Instructions will typically be written instructions on a package insert, a label, and / or on other components of the kit.

[0144] Depending on the type of assay performed, the kit may also include a device for either manipulating the device or (e.g., a device with one or a plurality of wells for performing a reaction). The device may include be a dropper, a swab, a stick, or the nozzle or outlet of an atomizer or aerosol can for containing and delivery of reagents. The kit may contain multiple formulations of different quantities of the amino acid sequences . The kit may also contain multiple formulations of different reagents.

[0145] The present kits will also typically include means for packaging the individual kit components, i.e., the amino acid sequence, the MHC molecule or MHC molecules (if included), and the written instructions for use. Such packaging means may take the form of a cardboard or paper box, a plastic or foil pouch, etc. Methods

[0146] The disclosure relates to methods of culturing cells and manufacturing the amino acid sequences disclosed herein. In some embodiments, the disclosure relates to a method of culturing one or a plurality of cells comprising: (a) exposing the one or plurality of cells comprising one or a plurality of amino acids disclosed herein to a cell culture medium. In some embodiments, the method comprises a step of seeding from about 1,000 cells to about 100,000 cells prior to performing step (a). In some embodiments, the disclosure also relates to a method of culturing a cell disclosed herein comprising (a) exposing the one or plurality of cells to one or more cell culture mediums for a time period necessary to produce the amino acids disclosed herein. In someDOCKET NO. STFD-002-PCT PCT APPLICATION embodiments, the method of culturing comprises about 24 hours, about 48 hours, about 72 hours or 96 hours or more in culture.

[0147] The present disclosure also relates to methods of identifying a selective immunomodulator of activity comprising: a) contacting an antigen with an amino acid sequence disclosed herein or a composition comprising the amino acid sequence or sequences disclosed herein; b) measuring the association of the amino acid sequence to the antigen in the presence and absence of an MHC molecule, or variant thereof; and c) comparing the rate of association of the amino acid sequence to the antigen.

[0148] The present disclosure also relates to methods of identifying a selective modulator of an antigen comprising: a) contacting an antigen with an amino acid sequence disclosed herein in a known concentration; b) measuring the rate association of the amino acid sequence to the antigen in the presence or absence of an unknown compound; and c) comparing the rate of association of the amino acid sequence to the antigen in the presence of a MHC molecule to the rate of association of the amino acid sequence to the antigen in the absence of the MHC molecule.

[0149] The present disclosuredisclosure also relates to methods of identifying a selective modulator of activity of an antigen comprising: a) contacting an antigen with an amino acid sequence in a known concentration; b) measuring the binding affinity of the amino acid sequence to the antigen; and c) comparing the binding affinity of the amino acid sequence to the antigen to the binding affinity of the amino acid sequence to a compound known to bind or not to bind to the amino acid sequence.

[0150] The present disclosure also relates to methods of identifying a therapeutic target for a treatment, the method comprising: (a) exposing one or a plurality of amino acid sequences disclosed herein to a treatment candidate; (b) measuring the association between the amino acid sequence and the treatment candidate; (c) selecting the treatment candidate if the association between the amino acid sequence and the treatment candidate comprises an EC50 value of less than about 1 micromolar when step (a) is performed in a known volume of liquid and the amino acid sequence is solubilized in liquid phase. In some embodiments, the EC50 value of less than about 900 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 800 nM is considered a criterion for selecting a treatmentDOCKET NO. STFD-002-PCT PCT APPLICATION candidate. In some embodiments, the EC50 value of less than about 700 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 600 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 500 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 400 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 300 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 200 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 100 nM is considered a criterion for selecting a treatment candidate. In some embodiments, the EC50 value of less than about 50 nM is considered a criterion for selecting a treatment candidate.

[0151] In some embodiments, the method further comprises (c) performing a mass spectrometry analysis on a selected target for a treatment associated with the disorder. In some embodiments, the methods further comprise correlating the association between the amino acid sequence and the treatment candidate with the likelihood that the target candidate will elicit a therapeutic immune response in a subject. In some embodiments the method further comprises selecting the antigen sequence as a treatment candidate if the association is above a first threshold, then the causal agent is selected as a therapeutic target for the disorder treatment, and wherein if the association is below the first threshold, then the candidate is not selected as a therapeutic target for the disorder treatment. In some embodiments, the disorder is a pathogen infection or a hyperproliferative disorder, such as cancer.

[0152] The disclosure further relates to methods of identifying a therapeutic target for a pathogen treatment, the method comprising: (a) quantifying or determining an association between one or more of the amino acid sequences of disclosed herein with a pathogen treatment candidate; and (b) correlating the association with a likelihood that a pathogen treatment candidate is an immunotherapeutic treatment, wherein if the association is above a first threshold, then the pathogen treatment candidate is selected as a therapeutic target for the immunotherapeutic treatment, and wherein if the association is below the first threshold, then the pathogen treatment candidate is not selected as a therapeutic target for immunotherapeutic treatment. In someDOCKET NO. STFD-002-PCT PCT APPLICATION embodiments, the amino acid sequence disclosed herein is SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof, wherein, if the sequence has an X value, X is any natural or non-natural amino acid. In some embodiments, the X residues are capable of associating with the therapeutic target or therapeutic antigen sequence.

[0153] The disclosure further relates to methods of identifying a therapeutic antigen sequence for treating a disorder, the method comprising screening a candidate compound for binding with, or activity against a therapeutic target, wherein the therapeutic target was identified via a disclosed method.

[0154] The disclosure further relates to methods of identifying an interaction between a pathogen protein and a host protein, the method comprising: (a) identifying a first pathogen protein that co- localizes with a first host protein in one or a plurality of bioassays; (b) calculating an association corresponding to a pathogen protein and a host protein in the presence, absence or quantity of amino acid sequences disclosed herein; and (c) correlating the association with the likelihood that the protein-protein interaction modulates pathogenicity of the pathogen. In some embodiments, the first pathogen protein is any of the antigens disclosed in Table Y, or those antigens that are functional variants thereof. In some embodiments, the host protein is a MHC molecule or functional variant thereof. In some embodiments, the first pathogen protein is a viral antigen from a lentivirus.

[0155] The disclosure further relates to methods of identifying an interaction between a first protein and a second protein, wherein the first protein is associated with a disorder of a subject or an epitope associated with the immunogenicity of an antigen, the method comprising: (a) identifying a first protein that co-localizes with an amino acid sequence disclosed herein in one or a plurality of bioassays; (b) calculating an association corresponding to the first protein and the amino acid. In some embodiments, the method further comprises (c) correlating the association with the likelihood that protein-protein interaction modulates immunogenicity of an antigen upon which the epitope is based. In some embodiments, the method further comprises exposing the first and second protein to a MHC molecule or functional variant thereof. In some embodiments, the second protein or amino acid sequence is SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof, wherein, if the sequence has an X value, X is any natural or non-DOCKET NO. STFD-002-PCT PCT APPLICATION natural amino acid. In some embodiments, the X residues are capable of interacting with the first protein.

[0156] The present disclosure provides devices, methods, and systems involving production, isolation and physical interrogation of protein-protein interactions in microengineered configurations designed to optimize identification and binding of antigens to immune machinery. It is another object of the disclosure to provide a medium to high-throughput assay for the screening of binding properties of antigen sequences for the presence of immunotherapeutic epitope or epitopes for a disease or disorder, such as cancer or other hyperproliferative disorders. In some embodiments, the antigens are cells presenting an antigen, such as or known antigens that are used to treat clinical disease. In some embodiments, the antigens are any drugs or agents that are used to treat human disease such association between the amino acid sequence disclosed herein (and, optionally, an MHC molecule) can be compared to the association of the amino acid sequence in the presence of an epitope known to bind or known not to bind to the amino acid sequence, such that epitopes may be characterized as an immunotherapeutic candidate. In some embodiments, the amino acid sequence is or comprises SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441, wherein, if the sequence has an X value, X is any natural or non-natural amino acid. In some embodiments, the X residues are capable of interacting with the cell presenting the antigen.

[0157] The herein described binding domains, complexes, cells or cell lines can be can be used in a variety of contexts and applications, for example, and without limitation, for capturing and / or purification of amino acid sequence:antigen protein complexes and / or amino acid sequence:MHC molecule:antigen and / or amino acid sequence: MHC molecule, and in crystallization studies and high-resolution structural analysis of such complexes. It is thus one of the aims of the specification to identify the binding domain as described herein, to stabilize such protein complexes and further to use these binding domains as co-crystallization aids for MHC molecules to complex with amino acid sequences disclosed herein, or, in other words, to facilitate crystallogenesis of antigen:amino acid sequence protein complexes. Additionally, and / or alternatively, the binding domains and preferably cellular systems expressing the binding domains, as described herein, can be useful forDOCKET NO. STFD-002-PCT PCT APPLICATION other applications such as ligand screening, drug discovery, immunization, all of which will be described into further detail below.

[0158] Some aspects of the specification relates to a method of identifying a binding domain that is directed against and / or specifically binds to a complex comprising an amino acid sequence disclosed herein and an antigen. The binding domain described herein can be any non-naturally occurring molecule or part thereof (as defined hereinbefore) that is capable of specifically binding to a complex comprising an amino acid sequence disclosed herein and an antigen. The binding domains as described herein may be protein scaffolds. The term protein “scaffold “ refers generally to folding units that form structures, particularly protein or peptide structures, that comprise frameworks for the binding of another molecule, for instance a protein (See, e.g., Skerra (2000), for review). In some embodiments, the scaffold is or comprises SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441, wherein, if the sequence has an X value, X is any natural or non- natural amino acid. In some embodiments, the scaffold further comprises a CD3 binding domain. IN some embodiments, the binding domain is a functional fragment of an antibody. In some embodiments, the binding domain comprises a scFv fragment of an antibody. A binding domain can be derived from a naturally occurring molecule, e.g. from components of the innate or adaptive immune system, or it can be entirely artificially designed. A binding domain can be immunoglobulin-based or it can be based on domains present in proteins, including but limited to microbial proteins, protease inhibitors, toxins, fibronectin, lipocalins, single chain antiparallel coiled coil proteins or repeat motif proteins. Examples of binding domains which are known in the art include, but are not limited to: antibodies, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies, the variable domain derived from camelid heavy chain antibodies (VHH or nanobodies), the variable domain of the new antigen receptors derived from shark antibodies (VNAR), alphabodies, protein A, protein G, designed ankyrin-repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, engineered CH2 domains (nanoantibodies), peptides and proteins, lipopeptides (e.g. pepducins), DNA, and RNA (see, e.g, Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 2004; Koide etDOCKET NO. STFD-002-PCT PCT APPLICATION al., 1998; Dimitrov, 2009; Nygren et al. 2008; WO2010066740). Frequently, when generating a particular type of binding domain using selection methods, combinatorial libraries comprising a consensus or framework sequence containing randomized potential interaction residues are used to screen for binding to a molecule of interest, such as an epitope or antigen sequence. It is another object of the disclosure to provide a low, medium or high-throughput assay of for the screening of antigens or antigen fragments for high affinity association between the amino acids disclosed herein and the antigens or antigen fragments. In some embodiments, the methods comprise exposing the amino acids disclosed herein to an isolated protein or library of proteins associated with a therapeutic target such that antigens or immunoreactive epitopes of the therapeutic target can be identified for therapeutic purposes or further analysis. In the case of antigen binding to the amino acid proteins can be a MHC molecule and / or an antigen or antigen fragment simultaneously or in sequence, allowing a time period for the amino acid and the antigen to associate, measuring or detecting the strength of the protein-protein interaction, and characterizing the antigen or antigen fragment as a high binder or potential immunotherapeutic molecule if the strength of the binding as measured by EC50 is less than about 1 micromolar.

[0159] Methods of the disclosure include the step of performing assays to confirm or measure the association between one or a plurality of the amino acid sequences disclosed herein and a polypeptide, including antigen sequences. In some embodiments, the methods comprises a step of performing cryo-EM. Cryogenic electron microscopy, also known as electron cryomicroscopy (cryo-EM), is an electron microscopy (EM) technique applied on samples cooled to cryogenic temperatures and embedded in an environment of vitreous water. Cryo-EM is an emerging, computer vision-based approach to determine 3-dimensional (3D) macromolecular structure with sub-nanometer resolution. Cryo-EM is applicable to medium to large-sized molecules in their native state. This scope of applicability is in contrast to X-ray crystallography, which requires a crystal of the target molecule, which are often impossible to grow, or nuclear magnetic resonance (NMR) spectroscopy, which is limited to relatively small molecules. Cryo-EM has the potential to unveil the molecular and chemical nature of fundamental biology through the discovery of atomic structures of previously unknown biological structures, many of which have proven difficult or impossible to study by conventional structural biology techniques.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0160] In cryo-EM, molecules are embedded in a frozen-hydrated state, suspended across holes in a thin carbon film (R. Henderson, Q. Rev. Biophys.37, 3 (2004); and W. Chiu et al, Structure 13, 363 (2005)), and then imaged with a transmission electron microscope in the presence of coherent, high-energy electrons (10-50 eVA2). A large number of such samples are obtained, each of which provides a micrograph containing hundreds of visible, individual molecules. In a process known as particle picking, individual molecules are imaged, resulting in a stack of cropped images of the molecule (referred to as particle images). Each particle image provides a noisy view of the molecule with an unknown pose. Once a large set of 2-dimensional (2D) electron microscope particle images of the molecule have been obtained, reconstruction is carried out to estimate the 3D density of a target molecule from the images. The ability of cryo-EM to resolve the structures of complex proteins depends on the techniques underlying the reconstruction process.

[0161] Generally, images obtained by cryo-EM can be analyzed to identify micrographs of single particles. Single particle selection can be done with the help of software tools such as SIGNATURE (Chen & Grigorieff (2007) J Struct Biol 157(1):168-73). The astigmatic defocus, specimen tilt axis, and tilt angle for each micrograph can be determined using the computer program CTFTILT (Mindell & Grigorieff (2003) J Struct Biol 142(3):334-47). Obtaining separate defocus values for each particle according to its coordinate in the original image improves the data quality of the cryo- EM density map which is obtained by averaging single-particle micrographs of particles.

[0162] Fitting of known atomic models within a cryo-EM density map is a common approach for building models of complex structures. A number of computational fitting tools are available which range from simple rigid-body localization of protein structures, such as Situs (Wriggers et al. (1999) J Struct Biol 125(2-3):185-95), Foldhunter (Jiang et al. (2001) J Mol Biol 308(5):1033-44 ) and Mod-EM (Topf et al. (2005) J Struct Biol 149(2):191-203), to complex and dynamic flexible fitting algorithms like NMFF (Tama et al. (2004) J Struct Biol 147(3):315-2), Flex-EM (Topf et al. (2008) Structure 16(2):295-307), MDFF (Trabuco et al. (2009) Methods 49(2):174-80) and DireX (Schroder et al. (2007) Structure 15(12):1630-41; Zhang et al. (2010) Nature 463(7279):379-83), which morph known structures to a density map. When an atomic model is not known, cryo-EM density maps can be used in building and / or evaluating structural models from a gallery of potential models that are constructed in silico (see Topf et al. (2005) J Struct BiolDOCKET NO. STFD-002-PCT PCT APPLICATION 149(2):191-203; Baker et al. (2006) PLoS Comput Biol 2(10):e146; DiMaio et al. (2009) J Mol Biol 392(1):181-90; Topf et al. (2006) J Mol Biol 357(5):1655-68; Zhu et al. (2010) J Mol Biol 397(3):835-51). A related template structure must be known for constrained comparative modeling or, for constrained ab initio modeling, the fold to be modelled must be relatively small. For example, an initial structure may be obtained using IMIRS (Liang et al. (2002) J Struct Biol 137(3):292-304). Further alignment and reconstruction can be performed with FREALIGN (Grigorieff (2007) J Struct Biol 157(1):117-25) using a known protein structure and a known structure of a heterologous protein or a close homologue as template.

[0163] Significant structural and functional information can be obtained directly from the density map itself. For example, at from about 5 to about 10 Å resolutions, some secondary structure elements are visible in cryo-EM density maps: α-helices appear as cylinders, while β-sheets appear as thin, curved plates. These secondary structure elements can be reliably identified and quantified using feature recognition tools to describe a protein structure or infer the function of individual proteins. At near-atomic resolutions (3-5 Å), the pitch of α-helices, separation of β-strands, as well as the densities that connect them, can be visualized unambiguously (see e.g., Cheng et al. (2010) J Mol Biol 397(3):852-63; Jiang et al. (2008) Nature 451(7182):1130-4; Ludtke et al. (2008) Structure 16(3):441-8; Yu et al. (2008) Nature 453(7193):415-9). The disclosure relates to a method comprising a step of creating a cryo-EM image or performing cryo-EM imaging comprising: (a) calculating an association between an amino acid sequence disclosed herein and a protein associated with a disorder or pathogen; (b) applying the association corresponding to a density map; (c) displaying an image of a protein on a display in operable communication with a controller or system comprising the computer program product. In some embodiments, the method further comprises (d) correlating the association with the likelihood that a protein-protein interaction is an immunotherapeutic candidate for a disorder. In some embodiments, the resulting image of the method of performing cryo-EM has a resolution from about 5 to about 20 angstroms, from about 5 to about 15 angstroms, or from about 5 to about 10 angstroms. In some embodiments, the image has a resolution of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 angstroms.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0164] Various other formats may be used to test for the presence, absence or quantity of a protein- protein interaction using the assay devices or system comprising one or a plurality of amino acid sequences disclosed herein. For instance, a “sandwich “ format typically involves mixing the test sample with probes conjugated with a specific binding member (e.g., antibody) for the analyte to form complexes between the analyte and the conjugated probes. These complexes are then allowed to contact a receptive material immobilized within the detection zone. Binding occurs between the analyte / probe conjugate complexes and the immobilized receptive material, thereby localizing “sandwich “ complexes that are detectable to indicate the presence of the analyte. This technique may be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich- type assays are described by U.S. Pat. No.4,168,146 to Grubb, et al. and U.S. Pat. No.4,366,241 to Tom, et al., which are incorporated herein in their entirety by reference thereto for all purposes. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an variant of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens or antigens, each hapten or antigen being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No.4,235,601 to Deutsch, et al., U.S. Pat. No.4,442,204 to Liotta, and U.S. Pat. No.5,208,535 to Buechler, et al., which are incorporated herein in their entirety by reference thereto for all purposes. Various other device configurations and / or assay formats are also described in U.S. Pat. No. 5,395,754 to Lambotte, et al.; U.S. Pat. No.5,670,381 to Jou, et al.; and U.S. Pat. No.6,194,220 to Malick, et al., which are incorporated herein in their entirety by reference thereto for all purposes.

[0165] Although various assay device configuration have been described herein, it should be understood that any known assay device may be utilized that is capable of incorporating an antigen and the amino acids of the disclosure. For example, electrochemical affinity assay devices may also be utilized, which detect an electrochemical reaction between a lysosomal protease (or complex thereof) and a capture ligand on an electrode strip. For example, various electrochemical assays and assay devices are described in U.S. Pat. No.5,508,171 to Walling, et al.; U.S. Pat. No. 5,534,132 to Vreeke, et al.; U.S. Pat. No.6,241,863 to Monbouquette; U.S. Pat. No.6,270,637 toDOCKET NO. STFD-002-PCT PCT APPLICATION Crismore, et al.; U.S. Pat. No.6,281,006 to Heller, et al.; and U.S. Pat. No.6,461,496 to Feldman, et al., which are incorporated herein in their entirety by reference thereto for all purposes.

[0166] One skilled in the art will readily appreciate the wide range of methods and techniques used for detecting the presence and / or quantity of protein-protein interactions in a complex sample. Techniques for detecting or quantifying protein-protein interactions include, but are not limited to, microscopy, immunostaining, immunoprecipitation, immunoelectrophoresis, Western blot, BCA assays, spectrophotometry, enzymatic assays, microchip assays, and mass spectrometry. In some embodiments, purification of proteins is necessary before detection or quantification techniques are employed. Techniques for purifying proteins include, but are not limited to, chromatography methods, including ion exchange, size-exclusion, and affinity chromatography, gel electrophoresis, magnetic beads comprising any antibody, antibody-like protein or antibody fragment or variant, Bradford protein assays. In some embodiments, methods of measuring the presence, absence, or quantity of association between an amino acid sequence disclosed herein and an unknown or known agent comprises performing any of the above-identified assays.

[0167] Provided herein is a method, such as an in vitro method, of capturing a complex comprising an amino acid sequence disclosed herein and an antigen comprising the steps of: (i) applying a solution comprising the amino acid sequences disclosed herein and one or a plurality of antigens (and, optionally, an MHC molecule) to a solid support; (ii) allowing a period of time sufficient to form a complex of a binding domain between the amino acid sequence and one of the plurality of antigens; and (iii) removing weakly bound or unbound molecules.

[0168] The disclosure relates to a method of purifying a complex comprising amino acid sequence disclosed herein and an antigen comprising the steps of: (i) applying a solution comprising the amino acid sequences disclosed herein and one or a plurality of antigens (and, optionally, an MHC molecule) to a solid support; (ii) allowing a period of time sufficient to form a complex of a binding domain between the amino acid sequence and one of the plurality of antigens; and (iii) isolating the complex of step (ii).

[0169] The above methods for capturing / purifying a complex comprising amino acid sequence disclosed herein and an antigen include, without limitation, affinity-based methods such as affinityDOCKET NO. STFD-002-PCT PCT APPLICATION chromatography, affinity purification, immunoprecipitation, protein detection, immunochemistry, surface-display, surface plasmon resonance, amongst others, and are all well-known in the art.

[0170] Crystallization and resolving the structure of a complex comprising amino acid sequence disclosed herein and an antigen. Crystallization of membrane proteins including antigens remains a formidable challenge. Although expression and purification methods are appearing that allow for the generation of milligram quantities, achieving stability with these molecules is perhaps the most difficult hurdle to overcome. First, binding domains as described herein may increase the stability of detergent solubilized a complex comprising amino acid sequence disclosed herein and an antigen, protecting them from proteolytic degradation and / or aggregation and facilitating the purification and concentration of homogenous samples of correctly folded proteins. Persons of ordinary skill in the art will recognize that such samples are the preferred starting point for the generation of diffracting crystals.

[0171] Crystallization is another major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. Proteins may be crystallized in a typical sparse matrix screening experiment, in which precipitants, additives and protein concentration are sampled extensively, and supersaturation conditions suitable for nucleation and crystal growth can be identified for a particular protein. Related to the sparse matrix screening approach is to generate structural variation in the protein itself, for example by adding ligands that bind the protein, or by making different mutations, preferentially in surface residues of the target protein or by trying to crystallize different species orthologues of the target protein (Chang 1998). One unexpected finding is the usefulness of binding domains as described herein that specifically bind to a complex comprising amino acid sequence disclosed herein and an antigen to introduce a degree of structural variation upon binding while preserving the overall fold of the complex.

[0172] Because crystallization involves an unfavorable loss of conformational entropy in the molecule to be assembled in the crystal lattice, methods that reduce the conformational entropy of the target while still in solution should enhance the likelihood of crystallization by lowering theDOCKET NO. STFD-002-PCT PCT APPLICATION net entropic penalty of lattice formation. The ‘surface entropy reduction’ approach has proved to be highly effective (Derewenda 2004). Likewise, binding partners such as ions, small molecule ligands, and peptides can reduce the conformational heterogeneity by binding to and stabilizing a subset of conformational states of a protein. Although such binding partners are effective, not all proteins have a known binding partner, and even when a binding partner is known, its affinity, solubility, and chemical stability may not be compatible with crystallization trials. Therefore, it was surprisingly found that the amino acid sequences disclosed herein can be used as tools to increase the probability of obtaining well-ordered crystals by minimizing the conformational heterogeneity in the target complex comprising amino acid sequence disclosed herein and an antigen.

[0173] Crystallization of MHC molecules for high-resolution structural studies is particularly difficult because of the amphipathic surface of these membrane proteins. Embedded in the membrane bilayer, the contact sites of the protein with the acyl chains of the phospholipids are hydrophobic, whereas the polar surfaces are exposed to the polar head groups of the lipids and to the aqueous phases. To obtain well-ordered three-dimensional crystals - a prerequisite to X-ray structural analysis at high resolution – amino acids disclosed herein are solubilized with the help of detergents and purified as protein-detergent complexes. The detergent micelle covers the hydrophobic surface of the membrane protein in a belt-like manner. Although attractive interactions between the micelles might stabilize the crystal packing, these interactions do not lead to rigid crystal contacts. Because many membrane proteins, including MHC molecules contain relatively small or highly flexible hydrophilic domains, a strategy to increase the probability of getting well-ordered crystals is to enlarge the polar surface of the protein and / or to reduce their flexibility. The most physiologic approach is to use a native ligand. The MHC molecules described herein can be used to enlarge the polar surfaces of the amino acid sequences disclosed herein through binding of an antigen and / or an MHC molecule, supplementing the amount of protein surface that can facilitate primary contacts between molecules in the crystal lattice with the polar surfaces of the other binding components. Binding domains described herein can also reduce the flexibility of its extracellular regions to grow well-ordered crystals.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0174] The disclosure relates to amino acid sequences useful as tools to crystallize a complex comprising the amino acid sequence disclosed herein and an antigen, and eventually to solve a crystal structure. In some embodiments, the complex which is crystallized by making using of a binding domain described herein further comprises a receptor ligand, more specifically an agonist. In some embodiments, the complex is in an active state or conformation.

[0175] A complex comprising amino acid sequence disclosed herein, an MHC molecule and an antigen may be crystallized using any of a variety of specialized crystallization methods for membrane proteins may involve lipid-based methods. These embodiments are lipid-based methods that include adding lipid to the complex prior to crystallization. Such methods have previously been used to crystallize other membrane proteins. Many of these methods, including the lipidic cubic phase crystallization method and the bicelle crystallization method, exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method).

[0176] The specification also relates to the use of a binding domain as described herein to solve a structure of a complex comprising amino acid sequence disclosed herein and an antigen, and optionally further comprising an MHC molecule. “Solving the structure “ as used herein refers to determining the arrangement of atoms or the atomic coordinates of a protein, and is often done by a biophysical method, such as X-ray crystallography.

[0177] In x-ray crystallography, the diffraction data when properly assembled gives the amplitude of the 3D Fourier transform of the molecule’s electron density in the unit cell. If the phases are known, the electron density can be simply obtained by Fourier synthesis. For a protein complex, the success to derive phase information from molecular replacement (MR) alone is questionable when the fraction of proteins with a known structure (the search models) is low (less than 50% of the amino acid content) and / or when the crystals exhibit limited diffraction quality. While the combination of multiple isomorphous replacement (MIR) and MR phasing has proven successful for protein complexes, the requirement of producing a good heavy atom derivative is almost always problematic. Over the past decade, classical MIR approaches have generally been superseded by the use of anomalous dispersion data principally using selenomethionine (SeMet) incorporation (MAD or SAD). In fact, the anomalous experimental data using Se-edge energiesDOCKET NO. STFD-002-PCT PCT APPLICATION generally provide superior and less biased phase information compared with either MIR or model- based MR phasing data. Accordingly, provided herein is the use of a binding domain described herein for the phasing of a complex comprising amino acid sequence disclosed herein and an antigen by MR or MAD. In particular, immunoglobulin single variable domains, including nanobodies, generally express robustly and are suitable for SeMet incorporation.

[0178] In many cases, obtaining a diffraction-quality crystal is the chief barrier to solving its atomic-resolution structure. Thus, the herein described binding domains can be used to improve the diffraction quality of the crystals so that the crystal structure of the target complex can be solved.

[0179] The disclosure also provides a general tool to obtain crystals of protein complexes. In particular, agonist-bound a crystal complex comprising amino acid sequence disclosed herein and an antigen crystals may provide three-dimensional representations of the active states of antigen binding domain, or epitope, within the antigen. These structures will help clarify the conformational changes responsible for the protein-protein interaction, and lead to more precise mechanistic hypotheses and eventually new therapeutics. Given the conformational flexibility inherent to ligand-activated MHC molecules and the greater heterogeneity exhibited by agonist- bound receptors, stabilizing such a state is not easy. Thus, such efforts can benefit from the stabilization of a complex of an agonist-bound receptor conformation bound to its heterodimeric MHC by the addition of binding domains that are specific for such a complex. Especially suited are binding domains that bind to the amino acid sequence that forms part of such a complex scaffold, since these binding domains can be used as general tools to stabilize all of the protein- protein interaction.

[0180] The disclosure also encompasses a method of determining the crystal structure of a complex comprising amino acid sequence disclosed herein and an antigen, the method comprising (i) exposing one or a plurality of the amino acid sequences disclosed herein to an antigen, optionally in the presence of a MHC molecule, for a time period sufficient to form a complex; and (ii) crystallizing the complex formed in step (i). In the above method of determining the crystal structure, the complex comprising amino acid sequence disclosed herein and an antigen may further comprise a MHC molecule, or a functional variant thereof. Any allele or MHC variant mayDOCKET NO. STFD-002-PCT PCT APPLICATION form the complex and the MHC molecule may comprise two subunits whose non-covalent association form a protein configured to bind to the antigen and / or the amino acid sequence identified herein. In some embodiments, the amino acid disclosed herein is or comprises SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441, wherein, if the sequence has an X value, X is any natural or non-natural amino acid.

[0181] Determining of the crystal structure may be done by a biophysical method such as X-ray crystallography. The method may further comprise a step for obtaining the atomic coordinates of the crystal (as defined hereinbefore).

[0182] The disclosure relates to identification of compounds targeting a complex comprising amino acid sequence disclosed herein and an MHC molecule. In the process of compound screening, there is a requirement for faster, more effective, less expensive and especially information-rich screening assays that provide simultaneous information on various compound characteristics and their affects on various cellular pathways (i.e. efficacy, specificity, and toxicity). Thus, there is a need to quickly and inexpensively screen large numbers of compounds in order to identify new specific ligands of the amino acid sequence identified herein, preferably conformation specific ligands, which may be potential new immunoreactive antigens. The present specification solves this problem by providing binding domains that stabilize a GPCR:G protein complex in a functional conformational state, that can then be used as immunogen or selection reagent for screening in a variety of contexts. In some embodiments, the amino acid disclosed herein is or comprises SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441, wherein, if the sequence has an X value, X is any natural or non-natural amino acid.

[0183] In some embodiments, methods of measuring the presence, absence, or quantity of association between an amino acid sequence disclosed herein and an unknown or know agent comprises performing any of the above-identified assays and comparing the associationDOCKET NO. STFD-002-PCT PCT APPLICATION determined in the above-identified assays with the association determined by performing an experiment performed in the Examples.

[0184] It is another object of the present disclosure to provide methods of replicating, manipulating, modifying protein-protein interaction comprising exposing a cell expressing one or more of the amino acid sequences disclosed herein to one or more polypeptides. In some embodiments, the one or more polypeptides comprises an antigen and / or an MHC molecule, or a functional variant thereof. It is another object of the present disclosure to allow medium to high‐ throughput assay for the screening of a library of antigens or cells comprising an antigen or antigens for binding constants.

[0185] The disclosure further provides a method of modeling a three-dimensional structure as between two or more amino acid sequences, said method comprising: (a) exposing an amino acid sequence of the disclosure to a polypeptide sequence comprises one or more epitopes associated with a disease or disorder; (b) calculating an association constant corresponding to the binding affinity between the amino acid sequence and the polypeptide or antigen (c) correlating the association constant of the association between the polypeptide with the amino acid sequence of the disclosure with: (i) spatial positions of amino acid residues within the amino acid sequence; and (ii) spatial positions of amino acid residues within the polypeptide; and (d) mapping a protein- protein interaction as between the amino acid sequence and the polypeptide.

[0186] In some embodiments, the disclosed methods further comprise employing one or more traditional structural biology methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and electron microscopy (EM), such as cryogenic electron microscopy (cryo-EM), sequentially with any of the disclosed methods to validate the quantity of the association calculated by step (b).

[0187] In some embodiments, the disclosure relates to a method of identifying perturbations in a protein-protein interaction comprising: (a) mutating one or more nucleic acids in the genome of a cell; (b) isolating a protein encoded by the one or more nucleic acids; (c) analyzing the mutation by exposing the protein encoded by the nucleic acid to one or more amino acid sequences ; wherein the step of analyzing comprises comparing the phenotypic profile associated with the mutation with the phenotypic profile associated with one or more nucleic acids free of the mutation.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0188]

[0189] Pharmaceutical Compositions

[0190] The disclosure relates to pharmaceutical compositions comprising any amino acid sequence disclosed herein. In some embodiments, the disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of an amino acid sequence disclosed herein. In some embodiments, the disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of an amino acid sequence disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises one or a plurality of amino acid sequences incorporated within an antibody or antibody fragment.

[0191] The term “antibody “ as used herein refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one “light “ and one “heavy “ chain. The variable regions of each light / heavy chain pair form an antibody binding site. As used herein, a “targeted binding agent “ is an antibody, or binding fragment thereof, that preferentially binds to a target site. In some embodiments, the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site. In some embodiments, the targeted binding agent may be a monoclonal antibody and the target site may be an epitope. “Epitope “ refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody. “Binding fragments “ of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies. An antibody other than a “bispecific “ or “bifunctional “ antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counter-receptor when an excess of antibody reduces the quantity of receptor bound to counter-receptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitroDOCKET NO. STFD-002-PCT PCT APPLICATION competitive binding assay). An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the disclosure; exemplary fragments include Fv, Fab, Fab’, single stranded antibody (svFC), dimeric variable region (Diabody) and di- sulphide stabilized variable region (dsFv). As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that have related side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non- polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are an aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding function or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragmentsDOCKET NO. STFD-002-PCT PCT APPLICATION or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function.

[0192] The present disclosure provides for the use of an antibody or binding composition which specifically binds to a specified antigen disclosed herein (SARS, EBV, NY-ESO-1, etc.), in some embodiments the antibody specifically binds the analog derived from a mammalian polypeptide, e.g., a polypeptide derived from a primate, human, cat, dog, rat, or mouse. Antibodies can be raised to various analogs, including individual, polymorphic, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms or in their synthetic forms. Additionally, antibodies can be raised to the analogs in their inactive state or active state. Anti- idiotypic antibodies may also be used. A number of antigens (or immunogens) may be selected to produce antibodies specifically reactive with ligand or receptor proteins. Synthetic analogs may serve as an immunogen for the production of monoclonal or polyclonal antibodies. Such antibodies may be used as antagonists or agonists for their targets modulating the disease state associated with the naturally occurring proteins and analogs listed above. Synthetic polypeptides of the claimed disclosure may also be used either in pure or impure form. Synthetic peptides, made using the appropriate protein sequences, may also be used as an immunogen for the production of antibodies. Naturally folded or denatured material can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated, e.g., for subsequent use in immunoassays to measure the protein, or for immunopurification methods.

[0193] Methods of producing polyclonal antibodies are well known to those of skill in the art.

[0194] Typically, an immunogen, such as a purified variant or analog of the disclosure, is mixed with an adjuvant and animals are immunized with the mixture. The animal’s immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein of interest. For example, when appropriately high titers of antibody to the immunogen are obtained usually after repeated immunizations blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive toDOCKET NO. STFD-002-PCT PCT APPLICATION the protein can be performed if desired. See, e.g., Harlow and Lane; or Coligan. Immunization can also be performed through other methods, e.g., DNA vector immunization. See, e.g., Wang, et al. ( 1997) Virology 228:278-284.

[0195] Monoclonal antibodies may be obtained by various techniques familiar to researchers skilled in the art. Typically, spleen cells from an animal immunized with a desired analog are immortalized, commonly by fusion with a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol. 6:51 1 -519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al. (eds.1994 and periodic supplements) Cell and Tissue Culture: Laboratory Procedures, John Wiley and Sons, New York, N.Y. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al. (1989) Science 246: 1275- 128.

[0196] Antibodies or binding compositions, including binding fragments, single chain antibodies, Fv, Fab, single domain VH , disulfide-bridged Fv , single-chain Fv or F(ab’)2 fragments of antibodies, diabodies, and triabodies against predetermined fragments of the analogs can be raised by immunization of animals with analogs or conjugates of analogs or receptor proteins with carrier proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to analogs described herein. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, usually at least about 300 μΜ, typically at least about 10 μΜ, at least about 30 μΜ, at least about 10 μΜ, and at least about 3 μΜ or more. These antibodies can be screened for binding to the naturally occuring polypeptides upon which the analogs are derived.

[0197] In some instances, it is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and ClinicalDOCKET NO. STFD-002-PCT PCT APPLICATION Immunology, 4th ed., Lange Medical Publications, Los Altos, Calif and references cited therein; Harlow and Lane ( 1988) Antibodies: A Laboratory Manual CSH Press; Goding ( 1986) Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y.; and particularly in Kohler and Milstein (1975) Nature 256:495-497, which discusses one method of generating monoclonal antibodies. Summarized briefly, this method involves injecting an animal with an analog described herein. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or “hybridoma “ that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the analog. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

[0198] Other suitable techniques involve selection of libraries of antibodies in phage or similar vectors. See, e.g., Huse, et ai. (1989) Science 246: 1275-1281 ; and Ward, et al. (1989) Nature 341 :544-546. The polypeptides and antibodies of the present disclosure may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see, Cabilly, U.S. Pat. No.4,816,567; and Queen, et al. (1989) Proc. Nat’ l Acad. Sci. USA 86: 10029-10033; or made in transgenic mice, see Mendez, et al. ( 1997) Nature Genetics 15: 146- 156; also see Abgenix and Medarex technologies.

[0199] The monoclonal antibodies and antigen-binding molecules of the present invention specifically include “chimeric “ antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is / are identical with or homologous to corresponding sequences inDOCKET NO. STFD-002-PCT PCT APPLICATION antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include “primitized “ antibodies comprising variable domain antigen- binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al, Proc. Natl. Acad. ScL U S A.81:6851 , 1985; Takeda et al, Nature 314:452, 1985, Cabilly et al, U.S. Patent No. 4,816,567; Boss et al, U.S. Patent No. 4,816,397; Tanaguchi et al, EP 0171496; EP 0173494; and GB 2177096.

[0200] An antibody, antigen-binding molecule, antibody fragment or antibody variant may also be modified by specific deletion of human T cell epitopes (a method called “deimmunization “) by the methods disclosed for example in WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). For detection of potential T cell epitopes, a computer modeling approach termed “peptide threading “ can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98 / 52976 and WO 00 / 34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. Human germline sequences are disclosed e.g. in Tomlinson, et al (1992) J. Mol. Biol. 227:776-798; Cook, G.P. et al. (1995) Immunol. Today Vol.16 (5): 237-242; and Tomlinson et al. (1995) EMBO J.14: 14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, for example as described in US Patent No.6,300,064. In some embodiments, theDOCKET NO. STFD-002-PCT PCT APPLICATION antibody or antibody fragment disclosed herein is or comprises SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27, SEQ ID NO:440 or SEQ ID NO 441, wherein, if the sequence has an X value, X is any natural or non- natural amino acid.

[0201] “Humanized “ antibodies, antigen-binding molecules, variants or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins of mostly human sequences, which contain (a) minimal sequence(s) derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, “humanized antibodies “ as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature, 321: 522-525 (1986); Reichmann et al, Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0202] Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; by Oi et al. (1986) BioTechniques 4:214; and by US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinantDOCKET NO. STFD-002-PCT PCT APPLICATION DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0203] Humanized antibodies may also be produced using transgenic animals such as mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a nonhuman CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

[0204] A humanized antibody can be optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al, Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al, Meth. Enzymok, 92: 3-16, 1982, and EP 239400).

[0205] The term “human antibody “, “human antigen-binding molecule “ and “human binding domain “ includes antibodies, antigen-binding molecules and binding domains having antibody regions such as variable and constant regions or domains which correspond substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) (loc. cit). The human antibodies, antigen-binding molecules or binding domains of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, in CDR3. The human antibodies, antigen-binding molecules or binding domains can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence. The definition of human antibodies, antigen-binding molecules and binding domains as used herein also contemplates fully human antibodies, which include only non-artificially and / or genetically altered human sequences of antibodies as those can be derived by using technologies or systems such as the Xenomouse. Preferably, a “fully humanDOCKET NO. STFD-002-PCT PCT APPLICATION antibody “ does not include amino acid residues not encoded by human germline immunoglobulin sequences.

[0206] In some embodiments, the antigen-binding molecules of the invention are “isolated “ or “substantially pure “ antigen-binding molecules. “Isolated “ or “substantially pure “, when used to describe the antigen-binding molecules disclosed herein, means an antigen-binding molecule that has been identified, separated and / or recovered from a component of its production environment. Preferably, the antigen-binding molecule is free or substantially free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antigen- binding molecules may e.g. constitute at least about 5%, or at least about 50% by weight of the total protein in a given sample. It is understood that the isolated protein may constitute from 5% to 99.9% by weight of the total protein content, depending on the circumstances. The polypeptide may be made at a significantly higher concentration through the use of an inducible promoter or high expression promoter, such that it is made at increased concentration levels. The definition includes the production of an antigen binding molecule in a wide variety of organisms and / or host cells that are known in the art. In preferred embodiments, the antigen-binding molecule will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated antigen-binding molecule will be prepared by at least one purification step.

[0207] The term “binding domain “ characterizes in connection with the present invention a domain which (specifically) binds to / interacts with / recognizes a given target epitope or a given target side on the target molecules (antigens), e.g. CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, and CD3, respectively. The structure and function of the first and / or second binding domain (recognizing CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM), and preferably also the structure and / or function of the effector binding domain (typically the third binding domain recognizing CD3), is / are based on the structure and / or functionDOCKET NO. STFD-002-PCT PCT APPLICATION of an antibody, e.g. of a full-length or whole immunoglobulin molecule, and / or is / are drawn from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or fragment thereof. Preferably the target cell surface antigen(s) binding domain(s) is / are characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). The effector (typically CD3) binding domain preferably also comprises the minimum structural requirements of an antibody which allow for the target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). It is envisaged that the first and / or second binding domain is produced by or obtainable by phage-display or library screening methods rather than by grafting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold.

[0208] According to the disclsoure, binding domains are in the form of one or more polypeptides. Such polypeptides may include proteinaceous parts and non-proteinaceous parts (e.g. chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise two or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids).

[0209] The disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of an antibody fragment comprising a first binding domain and a second binding domain, the first binding domain comprising an amino acid sequence specific for human CD3 and a second binding domain comprising an amino acid sequence disclosed herein or an antigen, specific for an antigen disclosed herein. In some embodiments, the antibody fragment is a single chain BiTE, organized as two humanized scFv amino acid sequences in single-chain form comprising a first domain and a second domain, wherein the first domain is a human CD3 binding domain and the second domain is an amino acid sequence disclosed herein, specific for an antigen. In some embodiments, the antibody fragment comprises one of the below sequences:

[0210]

[0211] MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGSGGGDOCKET NO. STFD-002-PCT PCT APPLICATION SGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGL EWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYG DSDWYFDVWGQGTLVTVSGGGGSLEVLFQGPDKEIAKEIFNMMFMLLWRVFRSQRIDA NNVELIKFNIRVLDWIMAEADNDLCYFIGTHDKCENPKEQWVANYQNLNNVVFTNKEL EDIYDLSNKEETKEVLKKFKEKVNQFYRHAFDIINKYGGGHHHHHH (SEQ ID NO: 351)

[0212]

[0213] SVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARMLLWRVERSYRIDKDNVE LIKFNIRVIDWIMAEAENDLCYFIGTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYD ESNKEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNV ELIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO: 30)

[0214] or an amino acid that comprises about 75%, 80%, 85%, 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one of the two above- identified amino acid sequences. In some embodiments, the antibody fragment comprises a first domain that comprises an anti-CD3 domain (a domain that targets and configured to bind to CD3), and a second domain comprising SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441 or a functional variant thereof that comprises that comprises about 75%, 80%, 85%, 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, or SEQ ID NO: 441. The instant disclosure is related to pharmaceutical compositions comprising the instant amino acid sequences or the pharmaceutical acceptable salts derived therefrom that comprise isotopes. In some embodiments, the compositions of the claimed disclosure may contain any isotope described in Cyr and Pearson (Stabilization of radiopharmaceutical compositions using hydrophilic thioethers and hydrophilic 6-hydroxy chromans. Cyr, John E.; Pearson, Daniel A. (Diatide, Inc., USA). PCT Int. Appl. (2002), WO 200260491 A2 20020808), which is herein incorporated by reference. In some embodiments the compositions of the disclosure comprise an analog or variant that comprises one or more of the following isotopes: 125I, , 311, 211At, 47Sc, 67Cu, 72Ga, 90Y, 153Sm, 159Gd, 165Dy, 166Ho, 175Yb, , 77Lu, 212Bi, 213Bi, 68Ga, 99Tc, 111In, 123I, and 3H. The pharmaceutical compositions of the instant disclosure or theDOCKET NO. STFD-002-PCT PCT APPLICATION pharmaceutical acceptable salts derived therefrom may be in a liquid or solid dosage form. Such compositions may include any type of dosage form such as tablets, capsules, powders, liquid formulations, delayed or sustained release, patches, snuffs, nasal sprays and the like. The formulations may additionally include other ingredients such as dyes, preservatives, buffers and anti-oxidants, for example. The physical form and content of the pharmaceutical formulations contemplated are conventional preparations that can be formulated by those skilled in the pharmaceutical formulation field and are based on well established principles and compositions described in, for example, Remington: The Science and Practice of Pharmacy, 19th Edition, 1995; British Pharmacopoeia 2000, each of which is incorporated herein by reference. The compositions of the present disclosure may also include other active agents useful in the treatment of cardiovascular conditions. Solid forms can be prepared according to any means suitable in the art. For example, capsules are prepared by mixing the analog composition with a suitable diluent and filling the proper amount of the mixture in capsules. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Diluents, but are not limited to, include various types of starch, cellulose, crystalline cellulose, microcrystalline cellulose, lactose, fructose, sucrose, mannitol or other sugar alcohols, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Non- limiting examples of tablet binders include, but are not limited to, starches, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including, but are not limited to, acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders. A lubricant can be used in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant include, but are not limited to, such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Tablets can be coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet. The compounds may also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established in the art. Also contemplated are liquid formulations and solid form preparations which are intended to beDOCKET NO. STFD-002-PCT PCT APPLICATION converted, shortly before use, to liquid form preparations. Such liquid forms include, but are not limited to, solutions, suspensions, syrups, slurries, and emulsions.

[0215] Liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats or oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g. , almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g. , methyl or propyl-p-hydroxy be nzoates or sorbic acid). These preparations may contain, in addition to the active agent, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The compositions may be in powder form for constitution with a suitable vehicle such as sterile water, saline solution, or alcohol, before use. Preparations may also contain mucosal enhancers.

[0216] In some embodiments, the oral transmucosal solid dosage further comprises a permeation enhancer. In some embodiments, the permeation enhancer is chosen from: a bile salt, sodium dodecyl sulfate, dimethyl sulfoxide, sodium lauryl sulfate, a derivative of a saturated or a unsaturated fatty acid, a surfactant, a bile salt analog, and a derivative of a bile salt. In some embodiments the oral transmucosal dosage form is chosen from: a chewing gum, a patch, a lozenge, a lozenge-on-a-handle, a tablet, a troche, a pastille, a sachet, a sublingual tablet, and a rapid disintegrating tablet. In some embodiments, the oral transmucosal solid dosage form of wherein the composition further comprises at least one flavoring agent, artificial coloring, sweetener, lubricating agent, disintegration agent, lubricating agent, diluent, base, or buffering agent. In some embodiments, the oral transmucosal solid dosage form further comprises a sustained release agent. The disclosure is directed to an oral transmucosal solid dosage form comprising from wherein the concentration of analog is from about 0.01 % to about 90% of the dry matter weight of the composition.

[0217] Solid dosage forms such as lozenges and tablets may also be used for oral transmucosal delivery of pharmaceuticals. For example, nitroglycerin sublingual tablets have been on the market for many years. The sublingual tablets are designed to deliver small amounts of the potent nitroglycerin, which is almost Immediately dissolved and absorbed. On the other hand mostDOCKET NO. STFD-002-PCT PCT APPLICATION lozenges or tablets are typically designed to dissolve in the mouth over a period of at least several minutes which allows extended dissolution of the lozenge and absorption of the drug.

[0218] Administration of lozenges or sublingual tablets generally utilize an “open “ delivery system, in which the drug delivery conditions are influenced by the conditions of the surrounding environment, such as rate of saliva secretion, pH of the saliva, or other conditions beyond the control of the formulation.

[0219] A lozenge-on-a-handle (similar to a lollipop) is another dosage form suitable for transmucosal drug delivery. In addition to being non-invasive and providing a particularly easy method of delivery, the lozenge-on-a-handle (or lozenge with an integrated oral transmucosal applicator) dosage form allows a patient or caregiver to move the dosage form in and out of the mouth to titrate the dose. This practice is called dose-to-effect, in which a patient or caregiver controls the administration of the dose until the expected therapeutic effect is achieved. This is particularly important for certain symptoms, such as pain, nausea, motion sickness, and premedication prior to anesthesia because each patient needs a different amount of medication to treat these symptoms. For these types of treatments, the patient is the only one who knows how much medication is enough. Once the appropriate amount of drug is delivered, the patient or caregiver can remove the lozenge-on-a-handle, thus, stopping delivery of the drug. This feature is especially important for particularly potent drugs, which may present a significant advantage of terminating drug administration once the desired effect is achieved.

[0220] As used herein, the term “oral transmucosal delivery “ (OTD) refers to the delivery of a pharmaceutical agent across a mucous membrane in the oral cavity, pharyngeal cavity, or esophagus, and may be contrasted, for example, with traditional oral delivery, in which absorption of the drug occurs in the intestines. Accordingly, routes of administration in which the pharmaceutical agent is absorbed through the buccal, sublingual, gingival, pharyngeal, and / or esophageal mucosa are all encompassed within “oral transmucosal delivery, “ as that term is used herein. Oral transmucosal delivery involves the administration of an oral transmucosal solid dosage form to the oral cavity of a patient, which is held in the oral cavity and dissolved, thereby releasing the pharmaceutical agent for oral transmucosal delivery. Of course, as the solid dosageDOCKET NO. STFD-002-PCT PCT APPLICATION form dissolves in the oral cavity, some of the saliva containing the pharmaceutical agent may be swallowed, and a portion of the drug may ultimately be absorbed from the intestines.

[0221] The compositions of the disclosure can be administered in a sustained release composition, such as those described in, for example, U.S. Pat. No.5,672,659 and U.S. Pat. Mo.5,595,760, and herein incorporate by reference. The use of immediate or sustained release compositions depends on the type of condition being treated.

[0222] The disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising: (i) an antibody fragment comprising a first binding domain and a second binding domain, the first binding domain comprising a sequence configured to bind to human CD3 or a fragment thereof, and a second domain comprising SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or a functional variant thereof that comprises that comprises about 75%, 80%, 85%, 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33; and (ii) a pharmaceutically acceptable carrier. In some embodiments, the antibody fragment is a bivalent amino acid sequence comprising a first scFv and a second scFV bound by a linker sequence. In some embodiments, the first scFv comprises an anti-human CD3 sequence and the second scFv comprises SEQ ID NO:31 or a functional variant thereof. In some embodiments, the first scFv comprises an anti-human CD3 sequence and the second scFv comprises SEQ ID NO:32 or a functional variant thereof. In some embodiments, the first scFv comprises an anti-human CD3 sequence and the second scFv comprises SEQ ID NO:33 or a functional variant thereof. In some embodiments, the first scFv comprises an anti-human CD3 sequence and the second scFv comprises SEQ ID NO:27 or a functional variant thereof. In each of the above sequences, if the antigen-binding region comprises an X within the formula, those X values may be any amino acid that binds an antigen of choice. In some embodiments, an embodiment comprises a scaffold region comprising from about 1 to about 3 alpha helices described herein and an antigen binding region, wherein the antigen binding region comprises SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 53) or a functional variant thereof that comprises at least about 88% sequence identity toDOCKET NO. STFD-002-PCT PCT APPLICATION SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 53).

[0223] In some embodiments, the pharmaceutical compositions of the disclosure comprises a therapeutically effective amount of a BiTE antibody fragment comprising a first scFv domain and a second scFv domain. In some embodiments, the first scFv domain comprises an anti-human CD3 amino acid sequence (or a sequence that target human CD3), and the second scFv domain comprises SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 53) or a functional variant thereof that comprises at least about 88% sequence identity to SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 52). Table W MHC Mol. Target TRACeR SEQ ID NO: Fragment A*02:01 PRAME SLLQHLIGL 70 A*02:01 NYESO1 SLLMWITQV 52 A*02:01 EBV CLGGLLTMV 352

[0224] In some embodiments, the second scFv domain further comprises SEQ ID NO:20 wherein the X amino acids are SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 53) or a functional variant thereof that comprises at least about 88% sequence identity to SLLQHLIGL (SEQ ID NO: 70), SLLMWITQV (SEQ ID NO: 52), or CLGGLLTMV (SEQ ID NO: 53). In the embodiment wherein the second scFv domain comprises an antigen binding domain specific for PRAME, the pharmaceutical composition may be administered to a subject in need thereof to treat cancer with cancer cells expressing PRAME. In the embodiment wherein the second scFv domain comprises an antigen binding domain specific for NY-ESO-1, the pharmaceutical composition may be administered to a subject in need thereof to treat cancer with cancer cells expressing NY-ESO-1. In the embodiment wherein the secondDOCKET NO. STFD-002-PCT PCT APPLICATION scFv domain comprises an antigen binding domain specific for EBV, the pharmaceutical composition may be administered to a subject in need thereof to treat Epstein Barr infection.

[0225] In some embodiments, the pharmaceutical composition comprises an antibody or antibody fragment that is bivalent and specific for human CD3 and specific for a viral antigen. In some embodiments, the disclosure relates to a method of treating a viral infection by administering a pharmaceutical composition comprising an an antibody or antibody fragment that is bivalent and specific for human CD3 and specific for a viral antigen disclosed herein.

[0226] The following Examples are intended to further illustrate certain characteristics of the disclosure not to be construed to limit the scope of the disclosure. Any references, patent publications or citations presented in this application are incorporated by reference in their entirety. References 1. Toor, J.S. et al. A Recurrent Mutation in Anaplastic Lymphoma Kinase with Distinct Neoepitope Conformations. Front Immunol 9 (2018). 2. Schumacher, T.N. & Schreiber, R.D. Neoantigens in cancer immunotherapy. Science 348, 69-74 (2015). 3. Croft, N.P. et al. Most viral peptides displayed by class I MHC on infected cells are immunogenic. P Natl Acad Sci USA 116, 3112-3117 (2019). 4. Neefjes, J., Jongsma, M.L., Paul, P. & Bakke, O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11, 823-836 (2011). 5. Pishesha, N., Harmand, T.J. & Ploegh, H.L. A guide to antigen processing and presentation. Nat Rev Immunol 22, 751-764 (2022). 6. 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TCR-engineered T cell therapy in solid tumors: State of the art and perspectives. Sci Adv 9, eadf3700 (2023). 19. Sharma, P. & Kranz, D.M. T Cell Receptors for Gene Transfer in Adoptive T Cell Therapy. Crit Rev Immunol 39, 105-122 (2019). 20. Fesnak, A.D., June, C.H. & Levine, B.L. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer 16, 566-581 (2016).DOCKET NO. STFD-002-PCT PCT APPLICATION 21. Lowe, K.L. et al. Novel TCR-based biologics: mobilising T cells to warm ‘cold’ tumours. Cancer Treat Rev 77, 35-43 (2019). 22. Boudousquie, C. et al. Polyfunctional response by ImmTAC (IMCgp100) redirected CD8(+) and CD4(+) T cells. Immunology 152, 425-438 (2017). 23. Yang, X. et al. Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy. Nat Biotechnol (2023). 24. Hennecke, J. & Wiley, D.C. T cell receptor-MHC interactions up close. Cell 104, 1-4 (2001). 25. Garcia, K.C. & Adams, E.J. How the T cell receptor sees antigen - A structural view. Cell 122, 333-336 (2005). 26. Holler, P.D., Chlewicki, L.K. & Kranz, D.M. TCRs with high affinity for foreign pMHC show self-reactivity. Nature Immunology 4, 55-62 (2003). 27. Linette, G.P. et al. Cardiovascular toxicity and titin cross-reactivity of affinity- enhanced T cells in myeloma and melanoma. Blood 122, 863-871 (2013). 28. Cameron, B.J. et al. Identification of a Titin-Derived HLA-A1-Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3-Directed T Cells. Sci Transl Med 5 (2013). 29. Sun, Y. et al. Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion. bioRxiv (2023). 30. Stewart-Jones, G. et al. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A 106, 5784-5788 (2009). 31. manuscript in preparation https: / / docs.google.com / document / d / 1LnqhfG75F00ZTIpQMcw29hIFBmJduhhud8MbZ OtxGGY / edit?usp=sharing. 32. Webb, A.I. et al. Functional and structural characteristics of NY-ESO-1-related HLA A2-restricted epitopes and the design of a novel immunogenic analogue. J Biol Chem 279, 23438-23446 (2004).DOCKET NO. STFD-002-PCT PCT APPLICATION 33. Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H.J. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res 33, W363- W367 (2005). 34. Cao, L.X. et al. Design of protein-binding proteins from the target structure alone. Nature 605, 551-+ (2022). 35. Huang, P.S. et al. RosettaRemodel: a generalized framework for flexible backbone protein design. PLoS One 6, e24109 (2011). 36. Lyskov, S. & Gray, J.J. The RosettaDock server for local proteinprotein docking. Nucleic Acids Res 36, W233-W238 (2008). 37. Bender, B.J. et al. Protocols for Molecular Modeling with Rosetta3 and RosettaScripts. Biochemistry-Us 55, 4748-4763 (2016). 38. Jacobs, T.M., Yumerefendi, H., Kuhlman, B. & Leaver-Fay, A. 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STFD-002-PCT PCT APPLICATION 45. Sun, Y. et al. Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes. Sci Adv 9 (2023). 46. Sun, Y. et al. Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes. Proc Natl Acad Sci U S A 120, e2304055120 (2023). 47. Coles, C.H. et al. TCRs with Distinct Specificity Profiles Use Different Binding Modes to Engage an Identical Peptide-HLA Complex. J Immunol 204, 1943-1953 (2020). 48. Venetz, N. et al. Application of the DARPin (R) technology for specific targeting of tumor-associated MHC class I:peptide complexes. Cancer Res 81 (2021). 49. Pei, R., Lee, J.H., Shih, N.J., Chen, M. & Terasaki, P.I. Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities. Transplantation 75, 43-49 (2003). 50. Wittenbrink, N. et al. 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[0227] Recently, we redesigned the superantigen Mycoplasma arthritidis Mitogen (MAM) into a peptide-focused pMHC-II binding platform as TRACeR-II31. TRACeR-II exhibits remarkable simplicity, stability and solubility in a non-immunogenic protein scaffold. We sought to exploit the potential of this small helical bundle-based structure (FIG.1B) to accommodate antigen-focused binding of the distinctively arched peptide conformation observed with MHC-I targets and the very small ligand footprint with MR1 targets. In the TRACeR-II design scheme, the N terminal flexible loop (residues 1-20), which we called the antigen recognition element (ARE), functions as the CDR equivalent to achieve specific target recognition. We hypothesized that a single ARE could also be used to discriminate the antigens in MHC-I or MR1, but the ARE’s critical placement would require a different binding mode, since TRACeR-II’s ARE is only compatible with the extended peptide conformation that MHC-II antigens adopt.

[0228] The adaptation to the different MHC family of structures is best optimized through experimental combinatorial library-based engineering (FIG. 1C), treating the structurally fluid ARE as a separate entity from the helical bundle scaffold. We used computational modeling to explore different docking orientations to guide the library design (workflow summarized in FIG. 1D). As a first target, we used the established HLA-A*02:01-bindng New York esophageal squamous cell carcinoma 1 antigen (NY-ESO-1, SLLMWITQV (SEQ ID NO: 52)). Starting from the available crystal structure to guide the modeling and design (PDB ID 1S9W32), we used a low-resolution docking algorithm, Patchdock33, to seed the search trajectories of a high resolution algorithm, RifDock34. This yielded a large number of compatible docking modes to the MHC structure. In contrast to TRACeR-II’s binding mode, with the helical bundle binding perpendicularDOCKET NO. STFD-002-PCT PCT APPLICATION to the antigen groove, RifDock models favored alternative orientations in which the helical bundle lies in parallel to the groove. We then carried out calculations to design the sequences to these models using an iterative protocol established in the Rosetta modeling suite35, 36, 37. The selected interface positions and their corresponding sequences were integrated to create a combinatorial library using Swiftlib38, which optimizes degenerate codon coverage by taking into account a given set of allowed amino acids at each position. At this stage, the library encompassed vast potential conformations, and we used yeast surface display to isolate a background sequence compatible with the MHC target (Table 1). The theoretical complexity for screening out initial binders in this background library was 1.3x1011, but our experimental screening coverage sampled a subset of the theoretical library with 1x109 transformed variants. Nonetheless, after 5 rounds of Fluorescence Activated Cell Sorting (FACS) against HLA-A*02:01 / NY-ESO-1, one single clone was enriched from the initial pool of a binding population, confirming our hypothesis that the platform based on the TRACeR-II scaffold could be engineered to bind MHC-I (FIG.9A).

[0229] After having the initial binder identified, we created a master library, holding constant the sequence in the scaffold but with maximally diversified combinatorial sequence in a subset of positions in the fluid ARE region to optimize binding to an expanded target repertoire. We randomized seven positions in the ARE, using NNK degenerate codons, with a combinatorial complexity of ~109, suitable to be fully covered by a yeast display screening strategy. To evaluate the possible space of pMHC antigens that can be specifically accessed by different ARE sequences, we screened the library against three divergent pMHC-I targets: HLA-A*02:01 / NY-ESO-1, HLA- A*02:01 / EBV LMP2 (CLGGLLTMV (SEQ ID NO: 53)), and HLA-B*08:01 / SARS-CoV-2 (HLRIAGHHL (SEQ ID NO: 54)). We include here MHCs of distinct A02 and B08 supertypes, defined as a grouping of alleles that share specific amino acid variants which define their peptide specificity. Four rounds of sorting were performed on each target followed by next-generation sequencing (NGS) to identify the most enriched clone. We achieved antigen-focused binding with all three targets with minimal cross-reactivity (FIG. 2A). These highly specific binders were isolated from the same master library without a negative selection step (FIG. 9B).). The entire process of isolating a binder can be performed in less than two weeks. This demonstrated the possibility for facile development of antigen-focused pMHC binders with the ability to targetDOCKET NO. STFD-002-PCT PCT APPLICATION polymorphic HLA supertype representatives — HLA-A*02:01 and HLA-B*08:01 — highlighting the generalizable nature of our TRACeR platform.

[0230] The design scheme for developing TRACeR-I platform can be extended to design binders against small molecule metabolites bound to MR1, which is structurally similar to MHC-I (FIG. 1D). The initial model was built based on the crystal structure of MR1 / 5-OP-RU (PDB ID 6PUC39). From computational modeling in steps one and two, we observed a very similar binding mode to MHC-I TRACeR, with the ARE region mostly contacting the metabolite. Given that the antigen represents a small exposed surface area that requires only a compact ARE region, we combined steps three and four into a single library. This combinatorial library incorporated diverse sequences on helical interface, while allowing all twenty amino acids on the ARE region. Focusing on 5-OPRU and 6-FP as representatives of the two main classes of ligands, we were able to enrich multiple clones from this single library that could differentiate the corresponding ligand / MR1 protein complexes (FIG.1D and FIG.9C).).

[0231] Biophysical characterization of TRACeR and binding profile evaluation

[0232] All TRACeRs developed are very specific to their cognate targets with minimum target cross-reactivity (FIG. 2A). As all TRACeRs share a very similar protein scaffold, we took the ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ as an example to evaluate the role of the ARE in binding MHC. Deleting the^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ completely abolished binding, confirming that the critical molecular surface requires both ARE and the helical bundle (FIG.10). Notably, the scaffold itself does not have detectable binding signals to the MHC. We have introduced a disulfide anchor to restrict the flexibility of the ARE, as the key mechanism to allow support of the diverse sequences in a mutagenic library. The presence of the disulfide is crucial, and mutating the involved cysteines to serine also abolished all binding signals on yeast (FIG. 10). However, because the disulfide crosslinks the N-terminal loop to a C-terminal helix, which can be challenging to form kinetically with certain loop conformations, we observed intermolecular disulfide cross-links while producing some of the constructs as soluble proteins. To address this issue, we introduced a circular permutation scheme that shifts the flexible region to an internal section of the protein, leading to a protein structure with different connectivity but similar overall 3D shape (FIG.11).DOCKET NO. STFD-002-PCT PCT APPLICATION

[0233] We chose our binder for HLA-A*02:01 / NY-ESO-1 as a model system to further investigate the TRACeR platform. NY-ESO-1 is a well-known cancer testis antigen, that is re-expressed in a variety of tumors and homogeneously expressed in the rare and aggressive soft-tissue cancers, myxoid round cell liposarcoma (94%) and synovial sarcoma (70%)40. Multiple TCRs and TCR- like antibodies have been developed for this target, allowing for a systematic comparison with our platform 30, 41, 42, 43.

[0234] We performed mutational scans on the NY-ESO-1 antigen peptide to define the breadth of specificity of our binder and obtain a low-resolution mapping of the ternary complex. We used our chaperone-mediated peptide exchange method to load individual antigen variants into HLA- A*02:01 conjugated to fluorescently labeled tetramers 44, 45, 46, and detected binding patterns to our TRACeR expressed on yeast by FACS. We first mutated each of the 7 non-anchor peptide residues to Ala, to define the contact footprint. We observed significantly reduced binding signals with the peptides carrying Ser1Ala, Met4Ala or Trp5Ala mutations while binding to other variants remained unchanged (FIG. 3A). This readout is consistent with the antigen conformation in the crystal structure of HLA-A*02:01 / NY-ESO-1 complex where residues 1, 4, 5 and 8 are the solvent- facing positions on the antigen. The sensitivity to alanine point mutations that result in complete loss of binding suggests that ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ must behave like a molecular imprinted binder, requiring multiple antigenbinding. Indeed, from the experiment in which we independently tested all other 19 possible mutations at each of the residues, we found that ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ essentially only binds to the cognate antigen with SLLMWITQV (SEQ ID NO:(FIG.3B). At position 1, we observed a weak cross-reactivity when the native Ser was substituted with Thr, suggesting a likely hydrogen bonding interaction to those residues. As a comparison, we also tested the specificity of an engineered TCR, 1G443, using our MHC-I tetramer library (FIG.3B), as well as comparing with reported specificity data on other NY-ESO- 1 binding TCRs47 and a DARPin48. These reported TCR and DARPin binders showed a broad range of cross-reactivity, especially at position 1. ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ shows an unprecedented specificity, suggesting a unique binding mode which can access the P1 position within the MHC- I groove in contrast to previously reported native or engineered receptors.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0235] We further assessed the HLA allelic cross-reactivity landscape of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ using single antigen beads (SABs). The beads are comprised of 97 HLA allotypes encoded with different colors 49, 50 and can be loaded with peptides of choice 45, 29. NY-ESO-1157-165 is expected to bind two types of the SABs carrying HLA-A*02:01 and HLA-A*02:06 in a similar conformation, based on NetMHCpan4.151 and HLA3DB predictions52 (FIG. 13A, FIG. 13B). By contrasting the binding signals with that of a non-binding variant of NY-ESO-1157-165 carrying the W5A substitution (NY-ESO-1W5A), we confirmed that the binding of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ to NY-ESO- 1157-165 is indeed peptide conformational specific, which is shared by the two MHCs, but the MHC’s peptide selectivity is also important to the overall specificity (FIG.3C).

[0236] The sensitivity of MHC-I TRACeR binding to position 1 of the peptide may also explain the unique metabolite sensitivity achieved with MR1 TRACeR. Comparing the structures of MHC- I and MR1, we observed that the small molecule metabolite in MR1 is located close to the peptide position 1 in MHC-I (FIG.3D). TRACeR’s apparent access to this region of the antigen pocket on MHC grants it a significant structural advantage, enabling the achievement of unparalleled antigen specificity.

[0237] To investigate the origin of the exquisite TRACeR specificity, we determined the co-crystal structure of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை^ି^^ଶ and HLA-A*02:01 / NY-ESO-1 at 2.3 Å resolution. In the asymmetric unit, ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ forms a domain-swapped dimeric structure interacting with two MHC complexes (FIG.4A, 4B). The dimer results from swapping two of the three helices in the scaffolding helical bundle. Despite the domain swapping, each half of the resulting helical bundle shows complete agreement with the equivalent regions in the wildtype MAM structure (FIGS. 14A and 14B). The two copies are near perpendicular to each other, with each half resembling two interlocking “U “-shaped clamps — the variable ARE region at the base turn of the “U “ and helical bundle portions as the arms. This spatial arrangement positions the ARE outwards, creating a cradling binding pocket for the antigen peptide in conjunction with the MHC facing residues on the scaffolding helical bundle (FIG. 4B, bottom). In ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ, the ARE loop adopts a helical structure that acts like a keystone in supporting the wedge between the stacking helices from TRACeR and MHC. The ARE also gains antigen access through a gapped opening in the MHC. This unique binding mechanism revealed by the structure offers a clearDOCKET NO. STFD-002-PCT PCT APPLICATION insight to the molecular basis of enhanced specificity: in contrast to cognate TCRs and engineered Abs, TRACeR makes complementary contacts throughout the entire length of the antigen (FIG. 4C, FIG.9C) We speculate that no TCR could reach a comparable level of specificity because of the limited range of interactions that can be made when the specificity depends on CDR3s. Indeed, superposition of known TCR:MHC complexes reveals that residues at both ends of the antigen are out-of-reach to TCRs53. This observation also agrees with our TCR antigen-selectivity data (FIG. 3B). Furthermore, while ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ has a larger binding footprint compared to TCRs (FIG. 16) most of the area coverage is on the antigen, leaving few and mostly knobs-into-holes contacts to the MHC helices (FIGS. 4C and 4D). As a result, ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ takes advantage of sidechain openings on the MHC where alanine and glycine are present. It uses the unique surface formed by the ridges in the long helices (from the ɑ1 and ɑ2 domains) on the MHC and the residue openings to dock on MHC in the antigen presentation face (FIG.4D).

[0238] Fifteen out of the twenty-four MHC interface residues are in direct contact with the ARE, and this is likely how ARE sequences can be adopted to different targets (Table 6). Because the antigen peptide in MHC-I adopts an arched conformation, interactions leading to antigen-focused recognition usually involve both side chain and backbone hydrogen bonding groups on the peptide. For NY-ESO-1157-165, four positions (P1, P4, P5, P8) have accessible sidechains, and every residue backbone except for positions 1, 7 and 9 has at least one available hydrogen bonding donor or acceptor to be accounted for (the P1, P7, P9 backbone amide and carbonyl groups are already hydrogen-bonded to MHC residues). In making the sidechain specific contacts, Asn76TRACeR is in the ARE responsible for the N-terminal side of the antigen; the hydrogen-bonding triad mediated by Asn76TRACeR unequivocally contributes to the selection in Ser1antigen. The same geometry tolerates Ser1Thrantigen substitution. For Met4antigen and Trp5antigen selectivity, several residues on TRACeR are involved in creating a hydrophobic pocking to accommodate the two hotspot residues: Phe13, Leu16, Trp17, Phe20, Tyr72, Phe80 and Tyr112. These interactions explain the mutagenesis results observed (FIG.15). For the interactions with the antigen backbone, P2 carbonyl is involved in a hydrogen-bonding network involving Asn76TRACeR; P3 amide is satisfied by Tyr72TRACeR; P4 amide by Tyr72TRACeR and carbonyl by Tyr112TRACeR; P5,DOCKET NO. STFD-002-PCT PCT APPLICATION P6 backbones are solvated; P8 amide by a water mediated hydrogen bond involving Asn10TRACeR (FIG.4E).

[0239] In the process of engineering TRACeR, we randomized the ARE and created mutations in the scaffolding helical bundle in successive combinatorial library optimization. The resulting binding conformation in the crystal structure agrees with our computational model in terms of the positioning of the helices relative to the MHC, but the domain-swapping was unexpected. Nonetheless, the domain-swapping is required to position the ARE in the proper location and the engineered disulfide and circular permutation are both required to stabilize the resulting dimer.

[0240] ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ outperformed other existing binders of the HLA-A*02:01 / NY-ESO-1 complex in binding specificity, implying that our binder would be relevant for therapeutic applications. To test this, we implemented our ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ as a BiTE, in which the NY- ESO-1 binder was fused to a humanized anti-CD3 single chain antibody fragment. We expressed ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 BiTE in insect cells and purified monomeric and dimeric fractions by size exclusion chromatography for testing in tumor cell killing assays (FIG.17). To test whether our ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 BiTE could elicit HLA-A*02:01 / NY-ESO-1 complex-specificseveral diffuse large B cell lymphoma cell lines expressing combinations of HLA-A*02:01 and / or NY-ESO-1. We co-cultured activated CD3+ human T cells (including both CD4+ and CD8+ cells) with fluorophore-labeled tumor cells and monitored their viability using a high-affinity nucleic acid stain. We observed remarkable killing specificity with the ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 BiTE, purified as a dimer, consistent with our x-ray structure. Killing was observed in both HBL-1 and HLY-1 cell lines which co-express both HLA-A*02:01 and NY-ESO-1 (FIG. 5B). In contrast, the ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 BiTE did not redirect T cells to kill SUDHL4 cells (HLA-A*02:01+ / NY-ESO-1-), nor SUDHL5 cells (HLA-A*02:01- / NY-ESO-1-) (FIG. 5B). The inability of T cells treated with ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 BiTEs to kill off-target SUDLH4 and SUDHL5 cells was not duemediated killing, as those cells were potently killed by antiCD19 / antiCD3 BiTEs (FIG. 5B). Thus, ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ BiTEs only enable killing of target cells when both cognate peptide and MHC are present. It has been recently reported that the efficacy of TCR-based BiTEs, or ImmTACs, areDOCKET NO. STFD-002-PCT PCT APPLICATION not affected by differences in affinity with the MHC target within the ranges between pM to ~600 nM54. Importantly, the nanomolar EC50 of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ falls in this relevant range of affinity. We then quantified the phenotype of T cells used in tumor killing assays and found that incubation with ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ results in a dose-dependent increase in frequency of T cells expressing CD69 and 4-1BB, indicating T cell activation (FIGS.5C and 5D). Our data demonstrate that TRACeRs in a BiTE format can confer highly specific target recognition and cell killing of tumor cells by T cells.

[0241] We further deployed our ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ as a chimeric antigen receptor (FIG.5E). To avoid tonic signaling by the dimeric of ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ, we redesigned the domain-swapped dimer into a monomer by rearranging the connections between the two helical bundle domains. The redesigned monomer is a stable protein that can be produced in high yields and maintains the binding specificity of the original domain-swapped version (See aforementioned Monomer Sequence and FIG.8).

[0242] Discussion

[0243] While there are around 60 targetable surface protein markers for solid tumors in CAR-T clinical trials to date55, the number of MHC-associated intracellular antigens is orders of magnitude greater56. Despite the growing pace of identifying MHC-presented antigens and their disease associations, without a general platform to recognition mechanism, the translational potential for these novel biomarkers remains unrealized.

[0244] We recognized that TCRs may not have been the right molecular solution to address this challenge, even though the conventional approaches have been largely relying on them. The molecular design of the TCR is indeed elegant; it has one set of loops to anchor on the MHC and another to interact with the antigen. This facilitates the two main functional requirements in the molecular recognition event, which are to bind specifically to MHCs at the right orientation and to assess the immunogenicity of the antigen. However, there are approximately 108 distinct TCR specificities in our bodies to respond to a much wider variety of foreign antigens57, all while having to maintain immunosurveillance and minimize autoimmunity. This requires TCRs to be promiscuous and of low affinity. Thus, the evolutionary forces that make TCR an excellent solutionDOCKET NO. STFD-002-PCT PCT APPLICATION for biology are the same ones making them extremely challenging to engineer into specific targeting molecules.

[0245] On a quest to an alternative molecular mechanism to recognize MHC in an antigen-focused fashion, we developed the TRACeR platform. The x-ray crystal structure for the TRACeR-MHC- I complex has uncovered a unique, novel binding mode, in which TRACeR can interact extensively with the presented antigen while only minimally engaging the MHC groove. The location of the ARE likely gives TRACeR-I its unprecedented and antigen-focused specificity profile. The binding pocket forms a cradle that spans the entire length of the antigen, with the ARE capping off the critical N-terminal side of the antigen. Importantly in comparison, most TCRs have no structural elements to reach and to discriminate mutations at the first antigen position. In all cases (MHC-I, MHC-II, MR1), a single stretch of amino acids is sufficient to define the target specificity. We currently rely heavily on library selection to identify the binding sequences, but with a clear view of the binding mechanism from the crystal structure, it is conceivable that in the future, a binder sequence could be directly designed in this highly constrained environment without experimental library screen.

[0246] While we achieved binding to different HLA alleles using the same scaffold sequence, the allelic compatibility is certainly not expected to be universal. HLA-C, for example, possesses few of the sidechain openings along the antigen groove that our current TRACeR-I relies on. Nonetheless, the relative ease to develop “antigen-focused “ binding TRACeRs is highly unusual and desirable. The crystal structure revealed a crisscross pattern of helices between MHC-I and TRACeR-I, and the intersection areas between the helices are relatively small in the overall contact surface, which makes scaffold-to-MHC interface compatible but not strongly reactive. Additionally, helices on TRACeR-I frame the antigen sidechains in the arched antigen conformation in MHC-I to render selectivity. Furthermore, the deletion of ARE also resulted in a complete loss of binding. These observations indicate that the compatibility of the scaffold to MHC requires the full complement of the specific conformation of the antigen and the complementary ARE. These features provide the remarkable allelic compatibility and antigen specificity.

[0247] As a proof-of-principle, we were able to achieve antigen-focused pMHC-I binding to three unrelated peptide targets with 7 randomized library positions (~108 complexity) and achieveDOCKET NO. STFD-002-PCT PCT APPLICATION ligand differentiation on MR1 with only 4 (~105 complexity). In contrast to the standard practice of using a native TCR as a starting point or using a synthetic combinatorial library of complexity ~1010 or greater10, these binders were created from scratch using a low complexity library while still show minimal non-specific binding. This strongly indicates the robustness of the TRACeR platform; this platform performs as well as TCRs in CAR-T killing assays, but offers also better specificity, ease-of-engineering and stability. We do not currently know the extent to which the platform can interact with the immense immunopeptidome, but for the characterized model target (NY-ESO-1), the isolated binder is exquisitely specific to the native antigen.

[0248] Through protein engineering, we were able to create a de novo platform for engineering new antigen-focused MHC binders that have exquisite specificity that surpasses natural TCRs. This opens the door to new ways to study and utilize the immunopeptidome and to expand the repertoire of targetable markers for a wide range of applications.

[0249] Methods

[0250] Library design, production and screening

[0251] Combinatorial DNA libraries for each design was constructed from assembly PCR using Ultramer oligonucleotides (Integrated DNA Technology) to encode the variable region. Library sequences were listed in Table 1). For library transformation, Saccharomyces cerevisiae yeast EBY100 cells were transformed with insert DNA and linearized pCTCON2 plasmid using an established protocol. After transformation, cells were grown overnight in SDCAA medium at 30 °C, passaged once, and stored in 20% glycerol solution at -80 °C.

[0252] Transformed yeast cells were grown in SDCAA media. For induction of expression, yeast cells were centrifuged at 2000g for 5 min and resuspended in SGCAA medium supplemented with 0.2% glucose at the cell density of 1e7 cells per ml and induced at 30 °C for 16-24 h. Cells were washed with PBSA (phosphate buffer saline (PBS) with 0.5% BSA) and labeled with pMHC tetramer / monomers, together with anti-c-myc fluorescein isothiocyanate (FITC, Miltenyi Biotech). After incubation for ~1 hr under room temperature, cells are washed twice and resuspended in PBSA, then run on Sony SH800 cell sorter. Library sorting results are shown in FIG.9A and 9B) NGS of final enriched pools were performed with Azenta Amplicon-EZ service.DOCKET NO. STFD-002-PCT PCT APPLICATION

[0253] Genes encoding the designed protein sequence were synthesized and cloned into pET- 24a(+) E.coli plasmid expression vectors (Genscript, C-terminal 6X His tag (SEQ ID NO: 353)). Plasmids were then transformed into chemically competent BL21(DE3) E.coli (ZYMO research). The cells are cultured in 2xYT media under 37 °C until OD reaches 0.6~0.8. Protein expression was then induced with 1 mM of isopropyl β-D-thiogalactopyranoside (IPTG) at 16 °C. After overnight expression, cells were collected and resuspended with 50mM Tris buffer (pH=8.0 300mM NaCl) and frozen at -80 °C until extraction and purification. The cell pellet was thawed and sonicated and purified by nickel affinity followed by size exclusion protein liquid chromatography (superdex 7510 / 300GL, GE Healthcare). All protein samples were characterized by SDS-PAGE. Protein concentrations were determined by absorbance at 280 nm measured with a Nanodrop spectrophotometer (Thermo Scientific) using predicted extinction coefficient.

[0254] CD spectra were measured on a JASCO CD spectrophotometer in a 1-mm pathlength cuvette (Hellma). Protein samples were at ~0.2 mg / mL in the 50 mM Tris buffer. Temperature melts were from 20 to 95 °C and monitored absorption signal at 222 nm in 1 °C increments per minute, with 10s of equilibration time and 1s digital integration time. Wavelength scans (200-260 nm) were collected at 20 °C and 95 °C, and again at 20 °C after fast refolding.

[0255] Biolayer interferometry binding data were collected on an Octet QK (ForteBio) and processed using the instrument’s integrated software. For binding assays, biotinylated pMHCs were loaded onto streptavidin-coated biosensors (ForteBio) at 1.25 ug / mL in the kinetic buffer (ForteBio) for 1800 s. Analyte proteins were diluted from concentrated stocks into the binding buffer. After baseline measurement in the binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing the target protein at the indicated concentration (association step) and then dipping the sensors back into baseline / buffer (dissociation). Association time is 1800 s and dissociation time is 1200 s, with a shaking speed of 1000 rpm. Data were analyzed and processed using ForteBio Data Analysis software 7.1.0.100. Example 2

[0256] Recombinant MHC protein expression, refolding, and purification with library screenDOCKET NO. STFD-002-PCT PCT APPLICATION

[0257] All peptide sequences are given as standard single letter codes. Peptides were purchased from Genscript at a purity of >90%. The placeholder peptide gTAX was purchased from Genscript at a purity of 98%, and the L-β-Phenylalanine (βF) containing placeholder peptide was synthesized in-house, as previously described. Peptides were solubilized in distilled water and centrifuged at 14000 rpm for 15 minutes. The concentration of each peptide solution was measured and calculated using the respective absorbance and extinction coefficient at 205 nm wavelength.

[0258] Plasmid DNA encoding the BirA Substrate Peptide (BSP, LHHILDAQKMVWNHR (SEQ ID NO: 354))-tagged luminal domain of the MHC-I heavy chain HLA-A*02:01 and human β2- microglobulin (β2m) were provided by the NIH tetramer facility (Emory University) and transformed into Escherichia coli BL21 (DE3) cells (Novagen). BSP-tagged MHC-I proteins were expressed in Luria-Broth media, and inclusion bodies were collected and purified using a standard protocol4. In vitro refolding of BSP-tagged pMHC-I molecules was performed by slowly diluting a 200 mg mixture of BSP-tagged MHC-I and hβ2m at 1:3 molar ratio over 24 hours in a refolding buffer (0.4 M L-Arginine, 100 mM Tris pH 8, 2 mM EDTA, 4.9 mM reduced glutathione, 0.57 mM oxidized glutathione) containing 10 mg of the placeholder peptide. BSP-tagged pMHC-I refolding was proceeded for 96 hours and followed by size-exclusion chromatography (SEC) for protein purification.

[0259] Biotinylation and tetramer library preparation of the HLA-A*02:01 proteins were performed as previously described1. The BSP-tagged proteins were biotinylated using the BirA biotin-protein ligase bulk reaction kit (Avidity) according to the manufacturer’s instructions and prepared at a final concentration of 2 mg / mL monomer. The level of biotinylation was evaluated by SDS-PAGE gel shift assay in the presence of excess streptavidin.

[0260] Biotinylated HLA-A*02:01 were then mixed with TAPBPR (10: 1 pMHC-I / TAPBPR molar ratio) and individual peptide from the NYESO alanine or position 1, 4, and 5 full scanned peptide libraries (1: 10 pMHC-I / peptide molar ratio). Each reaction was incubated 2 hours at room temperature (RT) and the peptide exchange reactions were confirmed by differential scanning fluorimetry. Meanwhile, Streptavidin-R-Phycoerythrin (Streptavidin-PE, Agilent Technologies, Inc.) at 4:1 monomer / streptavidin molar ratio was added to HLA-A*02:01 proteins in the presence of excess peptides over 10-time intervals every 10 mins at RT in the dark. Tetramerized moleculesDOCKET NO. STFD-002-PCT PCT APPLICATION upon peptide exchange were washed using Amicon Ultra centrifugal filter units with a 100 kDa membrane cut-off to remove excess peptide with a 1:1000 dilution of 1XPBS. Biotinylated MR1 proteins, which did not require peptide exchange, were prepared the same way as peptide exchanged molecules incubating the same amount of buffer. The resulting tetramers can be stored at 4°C for up to 4 weeks.

[0261] To screen for ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ cross-reactive interactions, 1 μM of soluble protein was mixed with 4 μL of beads (OneLambda Inc., CA, USA) that were pre-incubated over weekend (o / w) with either NY-ESO-1157-165 wild-type or W5A (3 μM), under agitation at 4oC. A variant of the molecular chaperone TAPBPR carrying the mutations S104F, K211L, and R270Q (TAPBPRFLQ (SEQ ID NO: 355) was used at 50 nM to facilitate peptide exchange during the o / w incubation45. Samples were incubated for 1 hour at 550 rpm, RT, washed three times followed by addition of the PE-conjugated anti-His tag antibody (Biolegend, 362603) for 30 min, 550 rpm at RT. After four washes, the levels of fluorescence intensity corresponding to the bound protein were measured using the Luminex 100 Liquid Array Analyzer System and the results were analyzed in GraphPad Prism v9.

[0262] Protein expression of the ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ-antiCD3 fusion in the Drosophila melanogaster S2 cell line was performed as previously described58. A DNA construct encoding the anti-CD3 fused with the ^^ ^^ ^^ ^^ ^^ ^^ெே^ுି^ாିௌூை,^ି^^ଶ His-tagged in the C terminus was cloned in pMT vector and the S2 cells were stably transfected. The cultures were induced with 1 mM CuSO4 and after 4 days, the supernatant was collected. The secreted protein was purified using a high-density metal affinity agarose resin (ABT, Madrid) and was further purified by SEC using a HiLoad 16 / 600 Superdex 200-pg column at a flow rate of 1 ml / min in 150 mM NaCl and 20 mM sodium phosphate buffer (pH 7.4). Example 3

[0263] Diffuse large B cell lymphoma culture

[0264] DLBCL cell lines were either directly received from the ATCC or verified by ATCC STR profiling. Cells were cultured at 37°C in a 5% CO2 incubator with Advanced RPMI (Gibco), 5% heat inactivated FBS (Gibco), Glutamax (Gibco), and penicillin / streptomycin (Gibco). Cells wereDOCKET NO. STFD-002-PCT PCT APPLICATION maintained between 200,000 – 1 million cells / mL and split every two days. Lines were routinely tested for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC).

[0265] For bispecific-induced cytotoxic assays, blood was collected from healthy, consenting donors by the Human Immunology Core at the University of Pennsylvania, and total CD3+ T cells were enriched by magnetic separation. Cells were cultured in Advanced RPMI (Gibco), 10% heat inactivated FBS (Gibco), Glutamax (Gibco), penicillin / streptomycin (Gibco), and 10mM HEPES (Quality Biological), supplemented with 300 U / mL recombinant IL-2 (NCI Biological Resources Branch). T cells were cultured at 1 million cells / mL and activated with a 1:1 ratio of Dynabeads Human T-Activator CD3 / CD28 beads (Gibco) for 72 hours, after which they were debeaded and expanded in IL-2-containing media.

[0266] Tumor cells were labeled with CellTrace CFSE (Invitrogen) at 1:1000 in PBS for 15 minutes at 37°C, following by extensive washout. Cells were counted and plated at 50,000 cells per well of a flat bottom 96-well plate. Primary human T cells were washed, and 100,000 T cells were plated in each well. Bispecific antibody stocks were made by serial dilution and spiked into each well as indicated by dose curves. Where indicated, CD19 / CD3 bispecific antibodies or CD19 / βGal antibodies were used as positive / negative controls (Invivogen). After ~18 hours of co- culture, Sytox Blue (Invitrogen) viability stain was added to each well. Cells were analyzed by the high throughput sampler of a LSR Fortessa (BD). Tumors were gated from immune cells, and the fraction live of each well was normalized to wells that received no antibody.

[0267] T cells incubated with off-target tumor cell lines SUDHL4 and SUDHL5 also displayed some activation in the presence of our TRACeR BiTE, albeit at a lower level (FIG.18A through 18D). This is likely due to activity of dimerized anti-CD3 scFv, which can facilitate cross-linking of CD3 on T cells and facilitate T cell activation independent of target recognition / killing. Example 4

[0268] Crystallography

[0269] HLA-A*2:01 / NY-ESO-1 / MAM-TRACeR complex was prepared by mixing MAM- TRACeR and HLA-A*2:01 / NY-ESO-1 at 1:1.3 molar ratio, mixture was incubated at 4 ºC for 1 hour. Unbound proteins were separated by size-exclusion chromatography using Superdex200DOCKET NO. STFD-002-PCT PCT APPLICATION increase 10 / 300 GL column, run at 0.5 mg / min flow rate with running buffer, 25 mM Tris pH 8.0 and 150 mM NaCl. Complex was concentrated to 10.4 mg / ml for crystallography, sample was mixed at 1:1 ratio with reservoir well solution and incubated at 20 ºC. Crystals were obtained in 200 mM Sodium sulfate and 20% w / v PEG 3350 using sitting drop vapor diffusion method. Crystals were harvested after transferring to cryogenic solution containing reservoir solution and 20% v / v Glycerol. Crystals were screened and data was collected on National Synchrotron Light Source (FMX, 17-ID-2) at Brookhaven National Laboratory (BNL) using Dectris Eiger 16M detector. Data was processed using XDS62 and structure of HLA-A*02:01 / NY-ESO-1 / MAM- TRACeR complex was solved by molecular replacement method using Molrep63 and Refmac564 software of ccp4 package suits65. Structure of HLA-A*02:01 / NY-ESO-1 (PDB ID, 1S9W)32 and model generated from AlphaFold structure prediction tool for MAM-TRACeR were used as search model. Complex structure was refined by Buster Global Phasing method 66 and Phenix 67 and COOT 68 was used for model building. Table 1. Summary of relevant amino acid sequences Description Amino acid sequence Note MKLRCENPKKA???N?QNLNNVVFTNKEL EDIYDLSNKEETKEVLKLFKLKVNQFYRH ? Are any AFGIVNDYGDKEIFNMMF??LW?VF?SQ??? amino acid Library 1: Redesign MHC-II binder ANNVE?IK?NI??LDWIMAEADNDLCYFIS and scanned interface for binding MHC-I Q (SEQ ID NO: 356) with library MKLRCENPK???????QNLNNVVFTNKELE DIYDLSNKEETKEVLKLFKLKVNQFYRHA ? are any FGIVNDYGDKEIFNMMFMLLWRVFRSQRI amino acid Library 2: Create library on ARE for DANNVELIKFNIRVLDWIMAEADNDLCYF and scanned selecting binders for multiple targets ISQ (SEQ ID NO: 357) with library MKLRCENPKKAYRDNAQNLNNVVFTNKELEDIYDLSN KEETKEVLKLFKLKVNQFYRHAFGIVNDYGDKEIFNM TRACeRNY-ESO-1,A02, from library 1 (initial MFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADN scaffold) DLCYFISQ (SEQ ID NO: 358) MKLRCENPKEQWVANYQNLNNVVFTNKELEDIYDLSN TRACeRNY-ESO-1,A02, from library 2 KEETKEVLKKFKEKVNQFYRHAFGIVNDYGDKEIFNMDOCKET NO. STFD-002-PCT PCT APPLICATION MFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADN DLCYFISQ (SEQ ID NO: 359) MKLRCENPKNLALVFSQNLNNFVVFTNKELEDIYDLSN KEETKEVLKKFKEKVNQFYRHAFGIVNDYGDKEIFNM MFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADN TRACeREBV,A02, from library 2 DLCYFISQ (SEQ ID NO: 360) MKLRCENPKNWCMYWWAQNLNNVVFTNKELEDIYDL SNKEETKEVLKKFKEKVNQFYRHAFGIVNDYGDKEIFN MMFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEA TRACeRSARS-CoV-2,B08, from library 2 DNDLCYFISQ (SEQ ID NO: 361) NVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFYRH AFGIVNDYGDKEIFNMMFMLLWRVFRSQRIDANNVELI TRACeRNY-ESO-1,A02, no loop KFNIRVLDWIMAEADNDLCYFISQ (SEQ ID NO: 36) MKLRSENPKEQWVANYQNLNNVVFTNKELEDIYDLSN KEETKEVLKKFKEKVNQFYRHAFGIVNDYGDKEIFNM MFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADN TRACeRNY-ESO-1,A02, no disulfide DLSYFISQ (SEQ ID NO: 362) DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRV LDWIMAEADNDLCYFIGTHDKCENPKNLALVFSQNLN NFVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFYR TRACeREBV,A02, Circular Permuted HAFDIINKYG (SEQ ID NO: 363) DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRV LDWIMAEADNDLCYFIGTHDKCENPKEQWVANYQNL NNVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFYR TRACeRNY-ESO-1,AO2, Circular Permuted HAFDIINKYG (SEQ ID NO: 364) MKLRCENP?KA????VSN?NNVVFTNKELE DIYDLSNKEETKEVLKLFKLKVNQFYRHA ? are library FGIVNDYGDKEIFNMMF??L??VFD?QRKE positions, Library 3: Redesign MHC-II binder ANNVEQIKRNIA?LD?IMA?ADNDLCYFIS position 12-15 interface for binding MR1 Q (SEQ ID NO: 365) is ARE MKLRCENPTKAFPRWVSNFNNVVFTNKELEDIYDLSNK EETKEVLKLFKLKVNQFYRHAFGIVNDYGDKEIFNMMF FMLRIVFDMQRKEANNVEQIKRNIAILDDIMAQADNDL TRACeR5-OP-RU,MR1CYFISQ (SEQ ID NO: 366) MKLRCENPKKAPFWGVSNFNNVVFTNKELEDIYDLSN KEETKEVLKLFKLKVNQFYRHAFGIVNDYGDKEIFNM MFMLLKGVFDMQRKEANNVEQIKRNIAILDDIMAQAD TRACeR6-FP,MR1NDLCYFISQ (SEQ ID NO: 367)DOCKET NO. STFD-002-PCT PCT APPLICATION DKEIAKEIFNMMFFMLRIVFDMQRKEANNVEQIKRNIAI LDDIMAQADNDLCYFIGTHDKCENPTKAFPRWVSNFN NVVFTNKELEDIYDLSNKEETKEVLKLFKLKVNQFYRH TRACeR5-OP-RU,MR1, Circular permuted AFDIINKYG (SEQ ID NO: 368) DKEIAKEIFNMMFMLLKGVFDMQRKEANNVEQIKRNIA ILDDIMAQADNDLCYFIGTHDKCENPKKAPFWGVSNFN NVVFTNKELEDIYDLSNKEETKEVLKLFKLKVNQFYRH TRACeR6-FP,MR1,Circular Permuted AFDIINKYG (SEQ ID NO: 369) MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISS LQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGSGGG SGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAAS GYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYN QKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCAR SGYYGDSDWYFDVWGQGTLVTVSGGGGSLEVLFQGP DKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRV LDWIMAEADNDLCYFIGTHDKCENPKEQWVANYQNL NNVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFYR TRACeRNY-ESO-1,A02-antiCD3 HAFDIINKYGGGHHHHHH (SEQ ID NO: 351) SVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARML LWRVERSYRIDKDNVELIKFNIRVIDWIMAEAENDLCYF IGTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDE SNKEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFN MMFMLLWRVFRSFRIDANNVELIKFNIRVLDWIMAEAD TRACeRNY-ESO-1,monomer NDLSYFISQ (SEQ ID NO: 30) Table 2. Binding kinetics summary of TRACeRs TRACeR Kd Kd error Kon Kon Kdis Kdis Cicular (M) error error permutation HLA-A02 / 2.17e-8 1.00e-9 9.77e2 1.86e0 2.12e-5 9.77e-7 Yes NY-ESO-1 HLA-A02 / 9.36e-8 6.44e-9 4.91e2 1.29e1 4.6e-5 2.92e-6 Yes EBV HLA-B08 / 3.71e-6 1.05e-7 3.34e2 8.89e0 1.24e-3 1.23e-5 No SARS-CoV-2 MR1 / 5OPRU 2.15e-7 1.25e-8 1.07e4 5.91e2 2.29e-3 3.99e-5 No MR1 / 6FP 6.1e-7 2.86e-8 3.66e2 9.82e0 2.23e-4 8.59e-6 NoDOCKET NO. STFD-002-PCT PCT APPLICATION Table 3. Alanine scan of NY-ESO-1 peptide and full scanned peptide library at position 1, 4, 5 SEQ Peptide EL-score EL_Rank BA-score BA_Rank Ave NB ID NO: 1370CLLMWITQV 0.3104 0.7334 0.7416 0.2327 0.3104 12371DLLMWITQV 0.2988 0.7663 0.4955 1.8331 0.2988 13372ELLMWITQV 0.5892 0.291 0.6362 0.6691 0.5892 14373FLLMWITQV 0.9034 0.0473 0.8913 0.0096 0.9034 15374GLLMWITQV 0.9034 0.0473 0.8355 0.0549 0.9034 16375HLLMWITQV 0.9128 0.0425 0.8245 0.0672 0.9128 17376ILLMWITQV 0.8859 0.0561 0.8536 0.0306 0.8859 18377KLLMWITQV 0.961 0.0183 0.8882 0.0099 0.961 19378LLLMWITQV 0.782 0.1189 0.8379 0.0529 0.782 110379MLLMWITQV 0.8203 0.0935 0.8811 0.0126 0.8203 111380NLLMWITQV 0.8713 0.0636 0.833 0.057 0.8713 112381PLLMWITQV 0.2468 0.9375 0.5779 1.0577 0.2468 113382QLLMWITQV 0.8586 0.0703 0.8117 0.0831 0.8586 114383RLLMWITQV 0.9331 0.0299 0.8723 0.0171 0.9331 115384TLLMWITQV 0.905 0.0466 0.8319 0.0578 0.905 116385VLLMWITQV 0.8781 0.0599 0.8304 0.0591 0.8781 117386WLLMWITQV 0.5341 0.3532 0.8235 0.0687 0.5341 118387YLLMWITQV 0.9385 0.028 0.9043 0.0085 0.9385 119388SLLCWITQV 0.8726 0.0629 0.9034 0.0086 0.8726 120389SLLDWITQV 0.9983 0.0007 0.9179 0.0073 0.9983 121390SLLEWITQV 0.9981 0.0008 0.9207 0.007 0.9981 122 391 SLLFWITQV 0.9467 0.025 0.8721 0.0173 0.9467 1 23392SLLGWITQV 0.9948 0.0022 0.9046 0.0084 0.9948 124393SLLHWITQV 0.9889 0.0046 0.8814 0.0125 0.9889 125394SLLIWITQV 0.9419 0.0267 0.8291 0.0604 0.9419 126 395 SLLKWITQV 0.9918 0.0034 0.8727 0.017 0.9918 1 27396SLLLWITQV 0.954 0.0216 0.8468 0.0385 0.954 128397SLLNWITQV 0.9918 0.0034 0.8934 0.0094 0.9918 129398SLLPWITQV 0.9977 0.001 0.8948 0.0093 0.9977 130 399 SLLQWITQV 0.9914 0.0036 0.8672 0.0207 0.9914 1 31400SLLRWITQV 0.9806 0.0...

Claims

DOCKET NO. STFD-002-PCT PCT APPLICATION CLAIMS We claim:

1. An amino acid sequence comprising a first scaffold region and an antigen binding region, wherein the amino acid sequence is from about 100 to about 120 amino acids in length; wherein the first scaffold region comprises, in an orientation from amino terminus to carboxy terminus, KEIFNMM (SEQ ID NO:1) and CX1X2X3X4X5X6X7X8C or CX1X2X3X4X5X6X7X8X9C (SEQ ID NO:20); and wherein X1through X9are any amino acid.

2. The amino acid of claim 1, wherein the first scaffold region comprises at least about 75% sequence identity to KEIFNMMFMLLWRVFRSQRIDAN (SEQ ID NO:2).

3. The amino acid sequence of claim 1 or claim 2, wherein the amino acid is about 122 amino acids in length.

4. The amino acid sequence of any of claims 1 through 3, wherein the antigen binding region is positioned after SEQ ID NO:3 in an orientation from amino terminus to carboxy terminus.

5. The amino acid sequence of claim 4, wherein the antigen binding region is positioned from about 3 to about 6 amino acids downstream from SEQ ID NO:3 in the carboxy terminal direction.

6. The amino acid sequence of any of claims 1 through 5, wherein the antigen binding region comprises at least about 7 contiguous amino acids in length and is variable in amino acid identity.

7. The amino acid sequence of any of claim 1 through 6, wherein the antigen binding region comprises EQWVANY (SEQ ID NO:6).DOCKET NO. STFD-002-PCT PCT APPLICATION 8. The amino acid sequence of any of claims 1 through 7, wherein at least five contiguous amino acids chosen from X1 through X9 comprise at least about 80% sequence identity to GTHDK (SEQ ID NO:7) of a functional variant thereof.

9. The amino acid sequence of any of claims 1 through 8 wherein the first scaffold region further comprises a contiguous amino acid sequence comprising at least about 75% sequence identity to NVELIKFNIRVLDWIMAEADNDLCYFI (SEQ ID NO:4) or a functional variant thereof.

10. The amino acid sequence of any of claims 1 through 9 further comprising a second scaffold region in the carboxy end of the sequence comprising an amino acid sequence, comprising at least about 75% sequence identity to KEETKEVLKKFKEKVNQFYRHAFDIINKYG (SEQ ID NO:5) or a functional variant thereof, from a amino terminal to carboxy terminal orientation 11. The amino acid sequence of any of claims 1 through 10 comprising an amino terminal sequence of DKEIA (SEQ ID NO:8) or a functional variant thereof that comprises at least about 80% sequence identity to SEQ ID NO:

8.

12. The amino acid sequence of any of claims 1 through 11, wherein a disulfide bond is position between the cysteines of SEQ ID NO:

3.

13. The amino acid sequence of any of claim 1 through 12, wherein the amino acid sequence is organized in threes alpha helices across a first and second scaffold region, wherein the first scaffold region comprises two alpha helices and the second scaffold region comprises an alpha helix and wherein the antigen binding domain comprises a loop.

14. A composition comprising any one or plurality of amino acid sequences of claims 1 through 13.DOCKET NO. STFD-002-PCT PCT APPLICATION 15. The composition of claim 14 comprising a dimer, wherein the dimer comprises two amino acid sequence chosen from one or a combination of the amino acid sequences of any of claims 1 through 13.

16. The composition of claim 15, wherein the dimer is a homodimer comprising amino acid sequences chosen from any of claims 1 through 13.

17. An amino acid sequence comprising a first scaffold region and a second scaffold with an antigen binding region positioned therebetween; wherein the first scaffold region and the second scaffold region comprise at least three alpha helices; and wherein the first and second scaffold regions are from about 20 to about 30 amino acids in length.

18. The amino acid sequence of claim 17, wherein the first and scaffold region comprise a first alpha helix comprising X1EEX2K , wherein X1 and X2 are independently selected from either V, K, T or I.

19. The amino acid sequence of any of claims 17 or 18, wherein the first and second scaffold region comprise a second alpha helix comprising MLLWR (SEQ ID NO: 9) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:

9.

20. The amino acid sequence of any of claims 17 or 18, wherein the first and second scaffold region comprise a third alpha helix comprising NVELIK (SEQ ID NO: 10) or a functional variant comprising at least about 80% sequence identity to SEQ ID NO:

10.

21. The amino acid sequence of any of claims 17 through 20, wherein the first scaffold region comprises on or a combination of: (i) SVEEIKKEYEERLKRFDEFVERILKETGN (SEQ ID NO:11) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:11;DOCKET NO. STFD-002-PCT PCT APPLICATION (ii) KEIANMARMLLWRVERSYRIDKDNVELIKFNIRVIDWIMAEAENDLCYFI (SEQ ID NO:51) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:51.; and (iii) NVELIKFNIRVIDWIMAEAENDLCYFI (SEQ ID NO:13) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:

13.

22. The amino acid sequence of any of claims 17 through 21, wherein the second scaffold region comprises on or a combination of: (i) KEETKEVLKKFKEKVNQFYEHAFDIINKYG (SEQ ID NO:14) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:14; (ii) DKEIFNMMFMLLWRVFRSFRIDAN (SEQ ID NO:15) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:15.; and (iii) NVELIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO:16) or a functional variant thereof that comprises at least about 75% sequence identity to SEQ ID NO:

16.

23. The amino acid sequence of any of claims 17 through 22, wherein the antigen binding domain comprises GTHDK (SEQ ID NO: 7) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:

7.

24. The amino acid sequence of any of claims 17 through 22, wherein the antigen binding domain comprises EQWVANY (SEQ ID NO: 6) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:

6.

25. The amino acid sequence of any of claims 17 through 22, wherein the antigen binding domain comprises GTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN (SEQ ID NO:18) or a functional variant comprising at least about 75% sequence identity to SEQ ID NO:

18.

26. The amino acid sequence of any of claims 17 through 25, wherein the amino acid comprises one or a combination of:DOCKET NO. STFD-002-PCT PCT APPLICATION (i) SEQ ID NO:11 or a functional variant thereof; (ii) SEQ ID NO:12 or a functional variant thereof; (iii) SEQ ID NO:13 or functional variant thereof; (iv) SEQ ID NO:14 or a functional variant thereof; (v) SEQ ID NO:15 or a functional variant thereof; (vi) SEQ ID NO:16 or functional variant thereof; (vii) SEQ ID NO: 18 or functional variant thereof; (viii) SEQ ID NO:440 or a functional variant thereof; and (ix) SEQ ID NO:441 or a functional variant.

27. The amino acid sequence of any of claims 17 through 26, wherein the sequence maintains a disulfide bond between the first scaffold and the antigen binding region.

28. A composition comprising one or a plurality of amino acid sequences of any of claims 17 through 27.

29. The composition of claim 28, wherein the amino acid sequence is associated with a second amino acid sequence.

30. The composition of claim 29, wherein the amino acid sequence is organized in a homodimer or single chain monomer.

31. A system comprising: (a) the amino acid sequence of any of claims 1 through 13 or 17 through 27; and (b) an antigen.

32. The system of claim 31, wherein the amino acid sequence is immobilized to a solid substrate.DOCKET NO. STFD-002-PCT PCT APPLICATION 33. A cell comprising a nucleic acid sequence encoding an amino acid sequence chosen from any of claims 1 through 13 or claims 17 through 27.

34. A kit comprising: (i) the amino acid sequence of any of claims 1 through 13 or 17 through 27; or a nucleic acid sequence encoding an amino acid sequence chosen from any of claims 1 through 13 or claims 17 through 27; and (ii) instructions for exposing the amino acid sequence to an antigen or an antigen fragment thereof.

35. A method of screening one or a plurality of antigens to identify an immunoreactive epitope comprising: (a) exposing the amino acid sequence of any of claims 1 through 13 or 17 through 27, or a cell expressing the same, to the one or plurality of antigens for a period of time sufficient to allow association of the amino acid sequence to the antigens; (b) measuring the association between the amino acid sequence and the antigen; (c) identifying the antigen amino acid sequence bound to the antigen binding region of the amino acid sequence.

36. The method of claim 35 further comprising the step of isolating the antigen after step (b).

37. The method of claim 35 or 36, wherein the step of identifying comprises performing protein sequencing.

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