Compositions and methods involving antibodies that bind to covalent peptide conjugates

Abstract

Compositions and methods are provided that include binding partners that specifically bind to target sites on proteins or peptides that include a covalently attached molecule. The binding partners are provided as antibodies and antibody derivatives that specifically bind to proteins and peptides that have been covalently modified by the attachment of a molecule, such as a drug. The binding partners are capable of specifically binding to covalently modified peptides presented in the context of the major histocompatibility complex (MHC). Uses and methods of the compositions for the prevention or treatment of disorders are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 154,627, filed February 26, 2021, and U.S. Provisional Patent Application No. 63 / 253,499, filed October 7, 2021, the entire disclosures of each of which are incorporated herein by reference.

[0002] [Statement Regarding Federally Sponsored Research] This invention was made with Government support under Grant Nos. CA194864, CA267362 and CA049152 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] [Sequence table] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on February 24, 2022, is named 58636_00482_SL.txt and is 212,998 bytes in size. Background

[0004] There is currently an unmet need for agents capable of binding to targets, including agents covalently attached to proteins or peptides, in particular to improve the efficacy of targeted therapies, as well as to improve tumor immunogenicity and the efficacy of immunotherapy against cancers driven by intracellular oncogenes. The present disclosure addresses these needs. Summary of the Invention

[0005] The present disclosure provides compositions and methods comprising binding partners that specifically bind to target sites on proteins or peptides that contain covalently attached molecules. This is believed to be the first disclosure of binding partners with such binding functionality. The present disclosure illustrates this approach using binding partners in the form of a number of antibodies and antibody derivatives that specifically bind to proteins and peptides that have been covalently modified by the attachment of molecules, exemplified by a variety of drugs. Furthermore, the present disclosure demonstrates that binding partners that specifically bind to peptides that have been covalently modified by the attachment of small molecule drugs are specific for said covalently modified peptides presented in the context of human leukocyte antigens (HLA), where HLA is a representative of the major histocompatibility complex (MHC). Thus, binding partners specific for peptide-drug conjugates in HLA complexes are presented. The present disclosure includes polynucleotides encoding the above binding partners, and cells modified to express the binding partners. The present disclosure includes diagnostic, prophylactic and therapeutic approaches using said binding partners. [Brief description of the drawings]

[0006] [Figure 1] Graph showing the results of phage enzyme-linked immunosorbent assay (ELISA) of phage-displayed antibody clones. G12C -GDP and KRAS G12C Binding to the -GDP-ARS-1620 conjugate was measured. For each clone, the bars in the graph are, from left to right, buffer, G12C-GDP-ARS, and G12C-GDP.

[0007] [Figure 2] KRAS in the GTPγS- or GDP-bound nucleotide state (with or without ARS-1620). G12C Graph showing binding of 12C-ARS FAB59 to IgG, characterized using a bead binding assay.

[0008] [Figure 3] 12C-ARS-Fab59 conjugated to ARS-1620 G12C -Graph showing specific binding to GDP.

[0009] [Figure 4] (A) shows data obtained using 12C-ARS-Fab59, which was shown to inhibit ARS-1620 / KRAS by pull-down assay of lysates prepared from cell lines. G12C The adducts were measured. (B) Shown are RAS-less MEFs (4A) and KCP (Kras G12C ; Tp53 R172H ;Pdx-Cre) mouse pancreatic cancer cells (4B) and immunoblots of 12C-ARS Fab-pulldown (PD) (treated with or without ARS-1620). (C) Shown are whole cell lysates and 12C-ARS Fab pulldowns (PD) from H358 and MIAPaCa-2 cells, treated as indicated. (D) Shown is ARS adduct formation in samples from C, quantified by LC / MS-MS assay. The concentrations of ARS-1620 and SHP099 were 10 μM for all panels.

[0010] [Figure 5] Data showing that 12C-ARS-Fab59 can be used to measure binding of ARS-1620 to mutant KRAS by pull-down assay using lysates prepared from animal tissues. Anti-pan RAS and anti-ERK2 (loading control) immunoblots of lysates and 12C-ARS Fab pull-down (PD). (A) LSL-KRAS G12C -Tp53 R270H (B) is LSL-KRAS G12C Tumors 3 days after oral administration of ARS-1620 (200 mg / kg / d) alone or in combination with the SHP2 inhibitor SHP099 (75 mg / kg / d).

[0011] [Figure 6] Binding of antibody clones to AMG510 conjugated with KRAS(G12C) peptide and poly-Ser peptide. For each antibody clone, the bars are from left to right: no target, KRAS(G12C), KRAS(G12C)-AMG, irrelevant peptide-AMG. The signals of the two negative controls, no target and KRAS(G12C) peptide not conjugated with drug, were too low to be visible on the graph. The antibody clones were displayed on the yeast cell surface, and binding of the target conjugated to fluorescently labeled streptavidin was detected by flow cytometry.

[0012] [Figure 7] Binding of the P2AMR-1 clone in human IgG1 format to AMG510 conjugated with KRAS(G12C) peptide (●) and the same peptide not conjugated with drug (■). The peptide was immobilized on streptavidin-coated beads, and the antibody bound to the beads was detected with a fluorescently labeled secondary antibody. D Values ​​are shown. Data shown are from triplicate determinations. Error bars are within the symbol sizes.

[0013] [Figure 8] Recognition of AMG510 presented on class I MHC molecules. AMG510-RAS(G12C) conjugate (●) and unconjugated peptide (■) were loaded onto HLA-A*03:01 and immobilized on streptavidin-coated beads. Antibody binding was detected in the same manner as in Figure 2. Apparent K D Values ​​are shown. Data shown here are from triplicate determinations. Error bars are within the symbol sizes.

[0014] [Figure 9] Schematic representation of the disclosed concept called HapImmune.

[0015] [Figure 10] Data showing the development of antibodies that bind to MHC / peptide-drug conjugate complexes. (A) Multiplex bead-binding assay (MBBA) of phages displaying different antibody clones. (B) MBBA assay of phages displaying different antibody clones (complex of BTK peptide conjugated with ibrutinib and HLA-A*01:01). (C) MBBA assay of phages displaying different antibody clones (EGFR peptide conjugated with osimertinib in complex with HLA-A*02:01).

[0016] [Figure 11] Binding titration graphs of purified antibodies targeting KRAS(G12C)-AMG510 conjugates using multiplex bead-binding assay (MBBA). Clone names are shown above each graph. Antigen nomenclature is as in Figure 10.

[0017] [Figure 12] Graph showing that antibody binding to HLA-complexed AMG510-peptide conjugates is not affected by the presence of free drug AMG510.

[0018] [Figure 13] Binding titration graphs of purified antibodies targeting BTK-ibrutinib conjugates using multiplex bead-binding assay (MBBA). Clone names are listed above each graph. Antigen nomenclature is as in Figure 10.

[0019] [Figure 14] Graph of binding titration of purified antibodies to KRAS(G12C)-AMG510 conjugates presented by endogenous HLA molecules on the cell surface. Raji cells were first incubated with KRAS(G12C)-AMG510 conjugates or KRAS (wild type) peptide, and excess conjugate and peptide were washed away. The amount of antibody detected using a fluorescently labeled secondary antibody is shown as a function of the IgG concentration used for staining. The apparent dissociation constant (K D ) values ​​were determined using nonlinear least-squares fitting of the 1:1 binding function. Data shown are from triplicate determinations.

[0020] [Figure 15] Antibody binding to KRAS(G12C)-expressing cell lines pretreated with AMG510. (A) Flow cytometry histogram. (B) Quantification of median fluorescence intensity of H358 cells treated with and without AMG510. (C) Quantification of median fluorescence intensity of H358 cells and HEK293T cells (negative control) treated with or without AMG510 and stained with AMRA3-7 antibody.

[0021] [Figure 16] Graph showing binding of P2AMR-1 IgG to cells preincubated with KRAS(G12C) peptide-AMG510 conjugate, KRAS(wild-type) peptide, or no peptide.

[0022]

[0033] FIG. 17 is a graph showing binding of purified antibodies in IgG format to the indicated drug-peptide / HLA complexes as measured using a multiplex bead binding assay (MBBA). (A) Antibody clones identified with AMG510 conjugated to the KRAS(G12C) peptide in complex with HLA-A*03:01 as antigen. (B) Antibody clones identified with AMG510 conjugated to the KRAS(G12C) peptide in complex with HLA-*11:01 as the antigen.

[0023] [Figure 18] Graph showing the cytotoxic effect of single-chain bispecific antibodies (scDb: single-chain diabody) on cells pulsed with exogenous peptide-drug conjugates. (A) Raji cells were first pulsed with AMG510 conjugated to a peptide corresponding to the KRAS(G12C) fragment or with a control peptide corresponding to KRAS(wild type). The pulsed cells were co-cultured with human T cells (effector:target=3:1) in the presence of single-chain bispecific antibodies (scDb) at the indicated concentrations. After incubation, dead cells were stained and detected by flow cytometry. Data shown here are from triplicate determinations. Error bars indicate s.d. If the error bars are not visible, the error is smaller than the symbol. (B) A comparable experiment with T2 cells and osimertinib conjugated to an EGFR peptide. As a negative control, the peptide was conjugated to beta-mercaptoethanol.

[0024] [Figure 19] Graph showing the specific cytotoxic effect of AMRA3-7_UCHT1 scDb against drug-treated lung cancer cell lines. (A) Lung cancer cell lines were treated with 100 nM AMG510 for 24 h and then co-cultured with human T cells (E:T=5:1) in the presence of AMRA3-7_UCHT1 scDb. After incubation, cell viability was measured. The scDb antibody showed a dose-dependent cytotoxic effect only against AMG510-treated cells carrying the cognate KRAS mutation (G12C) and HLA (HLA-A3). (B) Cytotoxic effect of scDb at 0.1 nM concentration. Data shown are from quadruplicate determinations.

[0025] [Figure 20] Binding titration curves of AMR-A3-7 and AMR-A3-7D displayed on the yeast cell surface. Binding to HLA-A*03:01 presenting AMG510 conjugated to the Cys residues of the 9-mer and 10-mer RAS(G12C) peptides, VVGACGVGK and VVVGACGVGK, respectively, is shown.

[0026] [Figure 21] Graph showing the cell-killing effect of AMRA3-7D scDb. (A) Dose-dependent cell killing effect of the AMRA3-7D scDb was first tested using Raji cells pulsed with AMG510 conjugated to a peptide corresponding to the KRAS(G12C) fragment or with a control peptide corresponding to KRAS(wild type). (B) Cell-killing effect of the AMRA3-7D scDb tested against H2122 non-small cell lung cancer cell line treated with AMG510 or DMSO (negative control) alone.

[0027] [Figure 22] Cell binding and killing effect of AMRA3-7D CrossMab. (A) Binding of AMRA3-7D to Jurkat cells expressing CD3 and to Raji cells not expressing CD3. (B) Dose-dependent cell killing effect of the AMRA3-7D CrossMab was first tested using Raji cells pulsed with AMG510 conjugated to a peptide corresponding to the KRAS(G12C) fragment or a control peptide corresponding to KRAS(wild type). (C) Cell killing effect of AMRA3-7D CrossMab tested using H2122 non-small cell lung cancer cell lines treated with AMG510 or DMSO (negative control) alone.

[0028] [Figure 23] Deep mutational scanning of AMR-A3-7D (a) Representative flow cytometry profiles of yeast cells displaying AMRA3-7D and its deep mutational scanning library population. (b) Mutation incidence at each position in a sorted subset of the deep mutational scanning library is shown in heat map format. Figure 23 discloses "GGWYPA" as SEQ ID NO: 155 and "ISYVKKLI" as SEQ ID NO: 153.

[0029] [Figure 24] Deep mutational scanning of OEA2-5 (a) Representative flow cytometry profiles of yeast cells displaying OEA2-5 in single-chain Fv format and its deep mutational scanning library population. (b) The mutation incidence at each position in a sorted subset of the deep mutational scanning library is shown in heat map format. Figure 24 discloses "EYVTMAL" as SEQ ID NO: 159 and "YSYWPI" as SEQ ID NO: 157.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] Every numerical range given throughout this specification includes its upper and lower limits, as well as every narrower numerical range that falls within that range, as if such narrower numerical ranges were all expressly written herein.

[0032] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When a range is so expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that by use of the antecedent "about," the particular value forms another embodiment. The term "about" with respect to numerical values ​​is arbitrary and may mean, for example, ±10%.

[0033] The present disclosure includes all amino acid sequences described herein and all nucleotide sequences encoding said amino acid sequences. All antibody sequences and antigen-binding fragments thereof are included. Polynucleotide and amino acid sequences having 80-99% similarity (including upper and lower limits and all numerical values ​​and numerical ranges therebetween) with the sequences provided herein are included in the present invention. All amino acid sequences described herein may include amino acid substitutions that do not adversely affect the function of the protein containing said amino acid sequence, such as conservative substitutions. In this regard, the present disclosure provides alternative residues for specific positions in the described binding partners, as described below. In certain instances, the alternative residues were identified by deep mutational scanning, which demonstrated the binding functionality of each binding partner containing the described amino acid changes. The present disclosure includes each binding partner with each alternative residue substituting only the original residue, and with any combination of the described alternative residues. Thus, the binding partners described herein can have any single residue change described or a combination of the described changes. Representative changes for specific antibodies are described in Tables A, B, C, and D. The alterations may be in CDR1, CDR2, CDR3, and combinations thereof. Alterations may also include amino acid insertions. The present disclosure includes each amino acid sequence encompassed by reference to the specific sequence identifiers and those listed in the preceding table, with the description of alternative amino acids.

[0034] As mentioned above, the present disclosure provides antibodies and antigen-binding fragments thereof (collectively "binding partners" and individually "binding partner"). The term "antibody" includes each form of binding partner herein. The binding partner specifically binds to a protein or fragment thereof, including a covalently attached molecule, or a peptide provided in peptide form. The covalently attached molecule forms a peptide conjugate. As used herein, "peptide conjugate" refers to any protein or peptide that has been modified to be covalently conjugated to another molecule. A peptide conjugate is considered a novel antigen, i.e., a neoantigen. The other molecule that is covalently conjugated to the protein or peptide to form the peptide conjugate is not particularly limited (unless the other molecule is an additional amino acid added to the peptide conjugate described). In embodiments, the molecule that is covalently conjugated to the protein or peptide may (or may have) have biological activity prior to conjugation, or may be biologically inactive prior to conjugation. In some embodiments, the molecule is a drug, including but not limited to a small molecule drug. Representative and non-limiting examples of drugs that are covalently attached to a peptide or protein to form a peptide conjugate are described below. Peptide conjugates include, but are not limited to, full-length proteins and fragments thereof that are covalently modified. Peptide conjugates include fragments of full-length proteins that contain covalent modifications, for example, produced by intracellular processing. In certain embodiments, full-length proteins can be covalently modified in cells and then processed to produce peptide conjugates that are fragments of the full-length protein. As described below, the produced peptide conjugates can be displayed on the cell surface.The cell surface display of the peptide conjugates may be any form of cell surface display, including but not limited to, by receptors having extracellular segments, or it may be displayed by any type of major histocompatibility complex (MHC) or human leukocyte antigen (HLA). Non-limiting examples of HLA types that display peptide conjugates to which the described binding partners bind with specificity are further described below.

[0035] In some embodiments, the binding partner preferentially binds to a protein or peptide, or a complex containing the protein or peptide, when covalently attached to a peptide conjugate, compared to the same protein or peptide not attached to an agent. Thus, the binding partners described herein do not detectably bind, or bind with reduced affinity, to the same protein or fragment thereof in the absence of the covalently attached molecule. In some embodiments, the binding partner binds to a protein or peptide containing a covalently attached agent with an affinity that is 10-10,000 times (including all values ​​and ranges of values ​​between 10-10,000) greater than the affinity for the protein or peptide without the covalently attached molecule. In this regard, without intending to be bound by any particular theory, it is believed that the presence of the covalently attached molecule contributes to the presence of an epitope to which the binding partner specifically binds.

[0036] In some embodiments, the molecule covalently conjugated to form the peptide conjugate is a drug and may be any targeted covalent inhibitor (TCI), but does not necessarily have to have inhibitory properties. In some embodiments, the molecule reacts with a specific residue in the target protein. In some embodiments, the molecule reacts at least partially with a segment of a protein or peptide that contains a nucleophilic or electrophilic residue. In some embodiments, the segment of the protein or peptide with which the molecule reacts includes any of Cys, Lys, Tyr, and His. In some embodiments, the molecule reacts at least partially with a segment of a protein or peptide that contains a wild-type Cys or a residue mutated to Cys, and thus may be covalently attached by a so-called sulfur tether. In embodiments, the agent is any agent described in Ghosh AK, Samanta I, Mondal A, Liu WR. Covalent Inhibition in Drug Discovery. ChemMedChem. 2019;14(9):889-906. doi:10.1002 / cmdc.201900107, De Cesco, et al., European Journal of Medicinal Chemistry 138 (2017) 96e114, or Bauer, RA, Drug Discovery Today, Volume 20, Number 9, September 2015, the disclosures of which relating to compounds that covalently modify protein targets are incorporated herein by reference.

[0037] In non-limiting embodiments, any of the amino acids Cys, Lys, Tyr, and His are present in the protein or peptide to which the molecule binds because the wild-type protein has been mutated to include one or a combination of the above residues. In non-limiting embodiments, the molecule binds to a protein or peptide that correlates with a pathology such as cancer. In some embodiments, the target (e.g., the protein or peptide to which the molecule covalently binds) is a receptor (including, but not limited to, any receptor having a catalytically active segment). In some embodiments, the agent binds to an enzyme that is not necessarily a receptor (including, but not limited to, any kinase). In some embodiments, the protein target includes a receptor with one or more activating mutations that promote ligand-independent enzymatic activity.

[0038] In some embodiments, the molecule targets and thus covalently binds to an amino acid sequence present in any of the following proteins and / or variants thereof: The protein and / or variants thereof may or may not contain a mutation, and may contain a mutation associated with a particular disease state, including, but not limited to, any type of cancer. In some embodiments, the protein is any protein described in "Visscher M, et al., Covalent targeting of acquired cysteines in cancer. Curr Opin Chem Biol. 2016;30:61-67. doi:10.1016 / j.cbpa.2015.11.004," the disclosure of which is incorporated herein by reference. Visscher et al. also teach a method to identify disease-associated mutant genes that introduce a Cys residue suitable for covalent modification. In some embodiments, the protein is KRAS, Bruton's tyrosine kinase (BTK), any member of the epidermal growth factor receptor (EGFR) family (also known as the ERBB family, including but not limited to EGFR (ERBB1), HER2 / NEU (ERBB2), HER3 (ERBB3), and HER4 (ERBB4); fibroblast growth factor receptor (FGFR); MET, BRAF, receptor kinases known in the art as cyclin-dependent kinases (CDK); Acetyl Choline Esterase (ACHE); TP53, IDH1, GNAS, FBXW7, CTNNB1, DNMT3A, any cathepsin (including cathepsin B, C, F, H, K, L, O, S, V, W, and X); any caspase; any protein involved in obesity (such as pancreatic lipase and METAP2), or any cancer-testis antigen.In embodiments, the agent targets and thus covalently binds to any viral protein (including, but not limited to, any viral DNA polymerase, RNA polymerase, reverse transcriptase, or polymerase, including RNA-dependent RNA polymerase, or any viral protein required for, for example, viral entry into a cell, or a protein encoded by any transposable element). In some embodiments, the agent targets EGFR and may be selected from PD168393, PF00299804 (dacomitinib), EKB569 (pelitinib), afatinib, WZ4002, osimertinib (formerly known as AZD9291), PF-06459988, nazartinib, naquotinib, olmutinib, avitinib, and rociletinib, neratinib, pyrotinib, poziotinib, and derivatives thereof. In some embodiments, the agent targets Bruton's tyrosine kinase (BTK) and may be selected from ibrutinib, acalabrutinib, zanubrutinib, CHMFL-BTK-11, ONO / GS-405, PRN1008, and CC-292. In some embodiments, the agent targets any p90 ribosomal S6 kinase (RSK) and may be selected from fluoromethylketone (FMK) and dimethyl fumarate. In some embodiments, the agent targets any FGFR and may be selected from FIIN-1, FIIN-2, FIIN-3, BGJ398, AZD4547, PRN1371, FGF401.

[0039] In a non-limiting embodiment, the binding partner specifically binds to a site contained in a neoantigen, which includes a covalently linked small molecule drug or other covalently linked molecule (as a component of an antigen associated with a particular MHC).

[0040] In some embodiments, the molecule that will be covalently linked to form the peptide conjugate targets any RAS oncogene protein product, including, but not limited to, HRAS, NRAS, KRAS4A, and KRAS4B. The amino acid sequences of RAS proteins are known in the art, and the numbering of the residues is the same for the relevant portions of all RAS isotypes discussed in this disclosure, and these amino acid sequences are available, for example, from UniProt P01116, which amino acid sequence is incorporated herein as of the effective filing date of this application or patent. The G12 position is numbered according to the known amino acid sequence, regardless of whether G12 is the 12th amino acid of the expressed RAS peptide sequence of this disclosure.

[0041] In one embodiment, the molecule covalently binds to a KRAS protein or peptide that contains a mutation. In embodiments, the mutation is at least one of KRAS residues 12, 13, or 61. References herein to agents include their names without regard to case.

[0042] In some embodiments, the agent targets a KRAS protein that includes a KRAS G12C mutation. In non-limiting embodiments, the agent that targets a KRAS protein is selected from 2E07, 6H05, SML-8-73-1, MRTX849, JNJ74699157, LY3499446, ARS-853, ARS-1620, MRTX1275, AMG510, or derivatives thereof. In some embodiments, the agent comprises a proteolysis targeting chimera (PROTAC) derivative of a covalent agent, a non-limiting description of which is provided in doi: 10.1021 / acscentsci.0c00411, which description of PROTACs is incorporated herein by reference. In some embodiments, the PROTAC is LC-1 or LC-2. In embodiments, the present disclosure relates to autophagy-mediated degraders (referred to as AUTACs), as described in doi.org / 10.1080 / 15548627.2020.1718362, the description of which is incorporated herein by reference.

[0043] A non-limiting example of a binding partner that binds to KRAS(G12C)-AMG510 is referred to herein as AMRA3-7D. The present disclosure includes all derivatives of AMRA3-7D described herein, including the alternative residues described below by deep mutational analysis, in the form of scDbs and CrossMab, representative amino acid sequences of which are provided. L ) and heavy chain (V H ) is as follows: TIFF2024513651000002.tif38169

[0044] A non-limiting example of a binding partner that binds to an epidermal growth factor receptor (EGFR)-osimertinib conjugate is referred to herein as OEA2-5. The disclosure includes all derivatives of OEA2-5 described herein, including alternative residues as described below by deep mutational analysis, in the form of a scDb, of which representative amino acid sequences are provided.

[0045] OEA2-5 light chain (V L ) and heavy chain (V H ) is as follows: TIFF2024513651000003.tif38169

[0046] In some embodiments, the binding partner binds to a protein in its native form, except that a drug or other molecule is covalently attached thereto. "Native form" refers to an intact protein that retains its biological function prior to the covalent attachment of a drug or other molecule. In some embodiments, a native form or protein is a form prior to fragmentation, such as by intracellular processing. Thus, in some embodiments, the binding protein binds to a full-length polypeptide that is covalently attached to a drug or other molecule, where the covalently attached drug or other molecule at least partially allows preferential binding of the binding partner. Generally, a polypeptide, as used interchangeably herein with the term "protein", comprises 50 or more contiguous amino acids. In some embodiments, the binding partner specifically binds to an intact protein that is covalently attached to a drug or other molecule. In other embodiments, the binding partner specifically binds to a peptide that comprises a covalently attached molecule. In some embodiments, the binding partner specifically binds to a peptide that has a particular amino acid sequence and is covalently conjugated to another molecule, such as a drug. In some embodiments, the binding partner preferentially binds to a peptide covalently attached to a molecule such as a drug, regardless of the sequence of the peptide. This preferential binding is in comparison to binding to the same peptide not conjugated to a drug. In some embodiments, the binding partner preferentially binds to a peptide containing a KRAS(G12) mutation, or a mutant thereof, where the mutant is at least 50% similar to a peptide containing KRAS(G12). This preferential binding is in comparison to binding to a KRAS(G12)-containing peptide or a mutant thereof, respectively, that is not covalently conjugated to a drug or other molecule.

[0047] In embodiments, the described binding partners bind with specificity to a peptide conjugate of suitable length for presentation at the major histocompatibility complex (MHC) (called human leukocyte antigen (HLA) in humans) or at the MHC or equivalent complex in non-human animals (including but not limited to non-human mammals).

[0048] Generally, peptide conjugates contain fewer than 50 contiguous amino acids. Thus, in embodiments, peptide conjugates containing the described epitopes can be 2-49 amino acids in length. In embodiments, the peptide to which the agent or other molecule is covalently attached, and the peptide to which the attached agent may be included in the epitope, contains 4-12 contiguous amino acids, which may or may not be derived from a longer protein during protein processing (e.g., an antigen processed for presentation by an MHC molecule). In embodiments, the agent is conjugated to a peptide that contains or consists of 7-30 amino acids. In embodiments, the agent or other molecule is conjugated to a peptide that contains or consists of 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids and may be presented in the context of MHC class I. In some embodiments, the agent or other molecule is conjugated to a peptide that is 9-30 amino acids (including the upper and lower limits and all values ​​and ranges of values ​​therebetween) and that can be presented in the context of MHC class II, hi some embodiments, the agent or other molecule is conjugated to a peptide that includes at least 7 amino acids.

[0049] In some embodiments, the binding partner specifically binds to a peptide conjugate that is covalently conjugated to a drug or other molecule, independent of MHC presentation. In some embodiments, non-limiting examples of which are described below in Example 3, the binding partner specifically binds to a peptide conjugate only if the peptide conjugate is presented by an MHC molecule. In some embodiments, the binding partner is capable of specifically binding to a peptide conjugate in both MHC-independent and MHC-presented contexts. In some embodiments, the MHC-peptide conjugate complex comprises an antigen to which the binding partner specifically binds.

[0050] In some embodiments, the binding partner can bind to cells via any MHC that can then present the peptide conjugate. In some embodiments, the HLA is expressed by cells that are restricted to class I, class II, or class III MHC presentation. In some embodiments, the binding partner can bind to cells that express class I MHC that presents the peptide conjugate. Those skilled in the art will recognize that class I MHC includes, among other things, polymorphic alpha chains and beta2 microglobulin, and that the peptide conjugate binds to the polymorphic chains.

[0051] In some embodiments, the cells are antigen-presenting cells (APCs). In some embodiments, the cells are so-called professional antigen-presenting cells, and thus may include, but are not limited to, macrophages and dendritic cells that display MHC class II. Those skilled in the art will recognize that MHC class II includes, among other things, the MHC polymorphic α and β chains, and the displayed peptide conjugates bind to both chains. In other embodiments, MHC class II may be displayed with peptide conjugates by other cell types (such as cancer / tumor cells), and thus the present disclosure provides for the direct recognition of such cells using the described binding partners, without the need for professional APCs.

[0052] In some embodiments, the peptide conjugates are displayed by non-classical MHC complexes, which may include CD1d, MR1, MHC-E, -F, -G and / or other emerging family members as will be recognized by those of skill in the art.

[0053] The present disclosure includes binding partners that bind with specificity to peptide conjugates displayed only by certain MHC types, and thus provide MHC type discrimination binding partners. Representative examples of such binding partners are described herein at least by FIG. 17.

[0054] In embodiments, the binding partners of the present disclosure are capable of binding with specificity to peptide conjugates comprising covalently conjugated agents or other molecules that are displayed by two or more specific MHC types. In embodiments, the binding partners of the present disclosure are capable of binding with specificity to peptide conjugates comprising covalently conjugated agents only in a specific MHC-associated context. In embodiments, the peptide conjugates are displayed by an MHC class I type selected from HLA-A, -B, -C, and combinations thereof. In certain aspects, the peptide conjugates are displayed in the context of any MHC class I that is A*02 / B*35 / C*04. In embodiments, the peptide conjugates are displayed by any of the class II MHCs that are DR*01 / DR*04 / DR*07 / DP*04. In some embodiments, the HLA includes A*01:01, A*02:01, A*03:01, A*11:01, A*24:02, A*26:01, B*07:02, B*08:01, B*27:05, B*39:01, B*40:01, B*58:01, or B*15:01. Specific examples of antibodies include those that bind to KRAS(G12C)-AMG510 conjugates presented by HLA-A*03:01 and HLA-A*11:01, BTK-ibrutinib conjugates presented by HLA-A*01:01, and EGFR-osimertinib conjugates presented by HLA-A*02:01. In non-limiting embodiments, the present disclosure provides scDbs specific for a particular agent covalently bound to a described peptide present on a particular HLA, or the same agent covalently bound to a described peptide present on two different HLAs. Representative scDbs are described in Example 4. Data obtained using the scDb are shown in Figures 18 and 19. Data obtained using the CrossMab format are shown in Example 5 and the accompanying figures.

[0055] In embodiments, the peptide conjugate is displayed by a cell that participates in a cell-mediated immune response or that can be a target of a cell-mediated immune response. In embodiments, the peptide conjugate, displayed in the context of any appropriate MHC, is included on a cell, which is recognized by a leukocyte, including but not limited to a T cell or a natural killer (NK) cell. In embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, a double positive CD4+ / CD8+ T cell, a CD4+ / CD8+ double negative T cell, or a γδ T cell. Thus, as described further below, the present disclosure provides binding partners configured to interact with both the presented peptide conjugate and a cell involved in a cell-mediated immune response. In embodiments, the particular described binding partner is capable of binding to a complex of a particular MHC (1) and a particular peptide conjugate (2). In embodiments, the particular described binding partner is capable of binding to a particular peptide conjugate presented by at least two different MHCs.

[0056] In embodiments, any binding partner of the present disclosure comprises at least one chain that comprises a complementary determining region (CDR), the CDR being a CDR1, CDR2, or CDR3 of any heavy or light chain amino acid sequence described herein. In certain examples herein, the CDRs are shown in bold. The amino acid sequences of the CDR sequences are separately encompassed in the present disclosure by their position in the described heavy and light chain amino acid sequences. The present disclosure includes binding partners that comprise the described heavy chain CDR1, CDR2, and CDR3. The present disclosure also includes binding partners that comprise the described light chain CDR1, CDR2, and CDR3. The present disclosure also includes binding partners that comprise the described heavy chain CDR1, CDR2, and CDR3 and the described light chain CDR1, CDR2, and CDR3. For amino acid sequences of the present disclosure that contain amino acids that include purification or protein production tags (such as a HIS tag and / or an AVI tag), the present disclosure includes the proviso that the sequences of the recited tags may be excluded from the amino acid sequence. Amino acids between the recited tags may also be excluded.

[0057] Binding partners of the present disclosure may be provided as intact immunoglobulins or as fragments of immunoglobulins, including, but not necessarily limited to, antigen binding (Fab) fragments, Fab' fragments, (Fab')2 fragments, Fd (N-terminal portion of the heavy chain) fragments, Fv fragments (two variable domains), diabodies (Dbs), dAb fragments, single domain fragments or single monomeric variable antibody domains, single chain diabodies (scDbs), isolated complementarity determining regions (CDRs), single chain variable fragments (scFvs), and other antibody fragments which retain antigen binding function. In some embodiments, one or more binding partners are provided as a component of a bi-specific T-cell engager (BiTE), a bispecific killer cell engager (BiKE), a CrossMab (e.g., a binding partner comprising four different chains; immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), or a chimeric antigen receptor (CAR) (e.g., for generating chimeric antigen receptor T cells (e.g., CAR T cells) and CAR natural killer (NK) cells, as well as killer macrophages). The present disclosure includes binding partners comprising the described heavy and light chain variable regions.

[0058] In some embodiments, the binding partner is multivalent. In some embodiments, a trispecific binding partner is provided. In some embodiments, the cell expresses at least a segment of one or more binding partners in the form of a CAR. In some embodiments, the binding partner of the present disclosure can be provided as a complex with a polynucleotide (such as an RNA polynucleotide) to form an aptamer. In some embodiments, the multivalent binding partner comprises one binding component, such as a paratope, that has specificity for a particular target on a desired cell type, such as any cancer cell marker. In some embodiments, a trispecific leukocyte engager is provided. In some embodiments, the binding partner may be a portion of a molecule that is activated only in the presence of a protease or other enzyme present in the tumor microenvironment, such an embodiment relates, for example, to a probody, examples of which are known in the art and described, for example, in doi: 10.1126 / scitranslmed.3006682, doi: 10.1038 / s41467-020-16838-w, and doi: 10.1038 / s41587-019-0135-x, the descriptions of which regarding probodies and protease activation are incorporated herein by reference. In one embodiment, the present disclosure provides a universal hapten that can be grafted onto an inhibitor.

[0059] In some embodiments, the CAR of the present disclosure comprises an scFv comprising a heavy chain and a light chain as described herein. As known in the art for previously reported CARs, the scFv is present in a contiguous polypeptide further comprising a CD3 zeta chain and a costimulatory domain. In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain or a CD28 costimulatory domain. The CAR may also comprise a co-receptor hinge sequence (such as a CD8 co-receptor hinge sequence).

[0060] In some embodiments, the binding partner of the present disclosure may include a constant region, such as an Fc region. Any isotype of constant region may be included. The binding partner that includes a constant region may be particularly suitable for antibody-dependent cell-mediated cytotoxicity (ADCC), and thus may function to kill target cells by cell-mediated responses by any of a variety of effector cells. Similarly, the constant region may be particularly suitable for enhancing complement-mediated responses.

[0061] In embodiments, the binding partners of the present disclosure may be modified to be present in a fusion protein. In embodiments, the antigen-binding segment of the binding partner may be present in a fusion protein and / or the constant region may be a component of the fusion protein. In embodiments, the fusion protein comprises amino acids from at least two different proteins. Fusion proteins can be produced using any of a wide variety of standard molecular biology approaches, including but not limited to expression from any suitable expression vector. In embodiments, the binding partners described herein may be present in a fusion protein with a detectable protein (e.g., green fluorescent protein (GFP), enhanced GFP (eGFP), mCherry, etc.). In embodiments, as an alternative to an expression vector, mRNA or chemically modified mRNA encoding any of the binding partners described herein can be delivered to a cell such that the binding partner is translated by the cell.

[0062] In some embodiments, the binding partners described herein are used to deliver drugs or toxins, and thus the binding partners may be provided as immunotoxins or in the form of antibody-drug conjugates (ADCs).

[0063] In embodiments, agents useful in the production of immunotoxins include enzymatically active toxins and enzymatically active fragments thereof. Suitable enzymatically active toxins include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. These can be provided as components of a fusion protein or can be covalently attached to the binding partner by any suitable conjugation method.

[0064] The binding partner may be linked to the chemotherapeutic agent using any suitable linker to form an antibody drug conjugate (ADC). In some embodiments, the linker comprises a disulfide, a hydrazine, or a thioether. The chemotherapeutic agent can be reversibly or irreversibly attached to the binding partner.

[0065] Cleavable linkers may be particularly useful for killing bystander cells. In some embodiments, a protease recognition site may be included to release the chemotherapeutic agent from the binding partner by the action of a protease that recognizes and cleaves the protease recognition site. Thus, the ADC may be considered to include a prodrug.

[0066] In some embodiments, the binding partner of the present disclosure may include a linking sequence. As a non-limiting example, the ScFv may include a linker that links together the paratope-containing segments. A suitable amino acid linker may be composed primarily of relatively small neutral amino acids such as glycine, serine, and alanine, and may include multiple copies of glycine and serine-rich sequences. In specific, non-limiting embodiments, the linker includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids. In one example, the linker may be a glycine-serine-alanine linker G4SA3 (SEQ ID NO: 11) or a glycine-serine linker (G4S)4 linker (SEQ ID NO: 12). In some embodiments, the binding partner may include a cellular localization signal, or a secretion signal. In some embodiments, the binding partner may include a transmembrane domain, and thus may be transported to and anchored in the cell membrane. For secretion, any suitable secretion signal can be used, many of which are known in the art.

[0067] In some embodiments, the binding partner may be part of an ADC, and thus the binding partner comprises a drug. The drug may include, but is not limited to, any suitable chemotherapeutic agent. In some embodiments, the ADC comprises a binding partner and a chemotherapeutic agent, such as a microtubule inhibitor, an alkylating agent, or a DNA minor groove binder. In some embodiments, the chemotherapeutic agent includes a maytansinoid, a dolastatin, an auristatin drug analog, or a cryptophycin. In some embodiments, the chemotherapeutic agent is a duocarmycin derivative, or an antibiotic, such as an enediyne antibiotic, or a pyrrolobenodiazepine (PBD), including dimers thereof. In some embodiments, the chemotherapeutic agent is an enzyme inhibitor, such as a topoisomerase or polymerase inhibitor. In some embodiments, the chemotherapeutic agent includes doxorubicin, or a metal-containing compound, such as a platinum-containing compound, non-limiting examples of which include cisplatin, carboplatin, or oxaliplatin. In embodiments, the ADC comprises a binding partner as described herein and any of the agents described in Barf and Kaptein, dx.doi.org / 10.1021 / jm3003203, J. Med. Chem. 2012, 55, 6243-6262 or Wilson et al., dx.doi.org / 10.1021 / jm400224q, J. Med. Chem. 2013, 56, 7463-7476 or Lambert and Morris, Adv Ther (2017) 34:1015-1035, the descriptions of which relating to agents used as components of such ADCs are incorporated herein by reference. In embodiments, the binding partner is conjugated to or otherwise includes a cytokine, thereby providing a cytokine conjugate. Cytokines include, but are not necessarily limited to, interleukins (including, but not necessarily limited to, IL-2 and IL-12), or interferons (IFNs).

[0068] Any suitable expression system may be used for the production of the binding partner. In general, a polynucleotide encoding the binding partner is used to express the binding partner in any suitable cell system, non-limiting examples of which include NS0 mouse myeloma cells, human cell lines, and Chinese hamster ovary (CHO) cells. In embodiments, the disclosure provides polynucleotides that can selectively hybridize to polynucleotides encoding any CDR or combination of CDRs described herein. In embodiments, the polynucleotides selectively hybridize to polynucleotides encoding the heavy chain CDR1, CDR2, and CDR3 of any of the binding partners described. In embodiments, the polynucleotides selectively hybridize to polynucleotides encoding the light chain CDR1, CDR2, and CDR3 of any of the binding partners described. In embodiments, the polynucleotides selectively hybridize to polynucleotides encoding the heavy and light chain CDR1, CDR2, and CDR3 of any of the binding partners described.

[0069] In embodiments, the binding partners described herein may be components of a fusion protein. In embodiments, for binding partners produced as, for example, fusion proteins, a peptide linker may be used. In embodiments, the peptide linker includes an optional self-cleaving signal. In embodiments, the self-cleaving signal may be present in the same open reading frame (ORF) as the binding partner. The self-cleaving amino acid sequence is typically about 18-22 amino acids in length. Any suitable sequence may be used, non-limiting examples of which include: T2A (EGRGSLLTCGDVEENPGP (SEQ ID NO: 7)); P2A (ATNFSLKQAGDVENPGP (SEQ ID NO:8)); E2A (QCTNYALKLAGDVESNPGP (SEQ ID NO:9)); F2A (VKQTLNFDLKLAGDVESNPGP (SEQ ID NO: 10)).

[0070] In cases where a segment of a protein that includes a binding partner described herein is a component of a library (including, but not limited to, a phage display library or a yeast surface display library), the disclosure includes the proviso that the binding partner may not include any segment of the library (including bacteriophage or yeast amino acid sequences, including, but not limited to, a phage coat protein or a yeast host protein, including, but not limited to, Aga2). Thus, in certain embodiments, the binding partner may be present in a fusion protein, but the fusion protein does not include a bacteriophage coat protein. In embodiments, any of the binding partners described herein may not include any pIII phage coat protein, or any portion of Ml, fd filamentous phage, T4, T7, or λ phage proteins.

[0071] In some embodiments, the binding partners of the present disclosure include a detectable label and can be used for diagnostic or therapeutic purposes. For example, the detectable label can be used to localize the binding partner for pathology and / or in vivo imaging approaches. In some embodiments, the binding partner is conjugated to any of a variety of radioactive agents, including, but not limited to, highly radioactive atoms (e.g., In111, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu). In certain embodiments, such as imaging, the binding partner may be conjugated to a radioactive atom (e.g., Tc99m (metastable technetium-99), 1123) for scintigraphy, or a spin label (e.g., 1123, 1131, 1124, F19, C13, N15, O17, or gadolinium (III) or manganese (II)) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, or "MRI"). In some embodiments, the radiopharmaceutical is suitable for use in CAT scans or PET imaging. In some embodiments, indium-111, technetium-99, or iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron-emitting labels such as fluorine-19 iodine-123 and iodine-124 can be used for positron emission tomography. Paramagnetic ions such as gadolinium (III) or manganese (II) can be used in magnetic resonance imaging MRI. In embodiments, the described radioisotopes attached to the described binding partners can also be used in therapeutic approaches. In embodiments, the radiopharmaceutical or isotope comprises an alpha-emitting radionuclide. In embodiments, the radiopharmaceutical or isotope comprises a beta-emitting radionuclide. In some embodiments, the present disclosure provides an antibody of the present technology conjugated to a diagnostic or therapeutic agent.Diagnostic agents may include radioactive or non-radioactive labels, imaging agents (e.g., for magnetic resonance imaging, computed tomography, ultrasound), and the radioactive labels may be gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotopes. Diagnostic agents are molecules that are administered conjugated to an antibody moiety (i.e., an antibody or antibody fragment or subfragment) and are useful in diagnosing or detecting disease by locating cells that contain an antigen.

[0072] Any of the binding partners described herein can be fully or partially humanized. Techniques for humanization of antibodies are known in the art and can be adapted for use in the present disclosure. In some embodiments, humanization can be performed, for example, by CDR-grafting. In some embodiments, one or more amino acids in the variable regions can be altered for humanization or to improve the properties of the binding partner. In some embodiments, one or more amino acids in the framework regions can be altered.

[0073] The present disclosure includes binding partners for use in diagnostic and therapeutic approaches. For therapeutic approaches, in certain embodiments, the binding partner may be delivered as an mRNA or DNA polynucleotide encoding the binding partner. Administering DNA or RNA encoding any of the binding partners described herein is also considered a method of delivering such binding partner to an individual or to one or more cells. Methods of delivering DNA and RNA encoding proteins are known in the art and can be adapted to deliver binding partners with the benefit of the present disclosure. In embodiments, one or more expression vectors are used (including viral vectors). Thus, in embodiments, viral expression vectors are used. The viral expression vectors may be used as naked polynucleotides or may include any viral particle, including but not limited to defective interfering particles or other replication-defective viral constructs, and virus-like particles. In embodiments, the expression vectors include modified viral polynucleotides derived from adenoviruses, herpes viruses, retroviruses, and the like. In some embodiments, retroviral vectors adapted from the murine Moloney leukemia virus (MLV) or lentiviral vectors may be used (e.g., lentiviral vectors adapted from human immunodeficiency virus type 1 (HIV-1)).

[0074] In one embodiment, oncolytic virus vectors are used. Oncolytic viruses (OV), including vaccinia (OVV), mediate anticancer effects by both direct tumor regression and stimulation of the innate immune response (increasing the production of type I interferon through the production of damage-associated molecular patterns (DAMPs) and the presence of viral-derived pathogen-associated molecular patterns (PAMPs)). In addition, OVV-mediated tumor regression may promote direct acquisition of tumor-derived antigens by host antigen-presenting cells in the tumor microenvironment, thereby leading to improved priming of T cells as well as coordination of the effector phase of the antitumor immune response. In another embodiment, recombinant adeno-associated virus (AAV) vectors can be used. In a particular embodiment, the expression vector is a self-complementary adeno-associated virus (scAAV).

[0075] Pharmaceutical formulations comprising binding partners are included in the present disclosure, which can be prepared by mixing with one or more pharma- ceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonicity agents, and the like. Carriers can be liquid, semi-solid, such as pastes, or solid carriers. Examples of carriers include water, saline or other buffers (phosphate buffer, citrate buffer, and the like), oils, alcohols, proteins (serum albumin, gelatin, and the like), carbohydrates (monosaccharides, disaccharides, other carbohydrates, and the like, including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, dextrins), gels, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents such as EDTA, salt-forming counterions such as sodium; non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG), or combinations thereof. In several embodiments, liposomal formulations comprising one or more binding partners are provided. Liposomal formulations include, but are not limited to, liposomal nanoparticles.

[0076] In some embodiments, an effective amount of one or more binding partners is administered to an individual in need thereof. In some embodiments, an effective amount is an amount that reduces one or more signs or symptoms of a disease and / or reduces the severity of the disease. An effective amount may also be an amount that inhibits or prevents the onset of a disease or the recurrence of a disease. The exact dosage can be selected by the individual physician in consideration of the patient to be treated. Dosage and administration can be adjusted to provide a sufficient level of the binding partner to maintain the desired effect. Additional factors that may be considered include the severity and type of disease state, the patient's age, weight, sex, desired duration of treatment, method of administration, time and frequency of administration, drug combinations, reaction sensitivities, and / or tolerance / response to treatment.

[0077] The binding partners and pharmaceutical compositions comprising the binding partners may be administered to an individual in need thereof using any suitable route, including, for example, intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, oral, topical, or inhalation, depending on the particular condition being treated. The compositions may be administered parenterally or enterally. The compositions may be administered as a single dose or as multiple doses, or may be administered continuously over a period of time. For example, administration may be a pre-specified number of doses, or daily, weekly, or monthly, which may be continuous or intermittent, as may be therapeutically indicated.

[0078] In some embodiments, the individual in need of the compositions of the present disclosure has been diagnosed with or is suspected of having cancer. In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma or other brain cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or other skin cancer, sarcoma (including but not limited to fibrosarcoma, hemangiosarcoma, osteosarcoma and rhabdomyosarcoma), any hematological cancer (including all types of leukemia, lymphoma and myeloma). In some embodiments, the individual is in need of treatment for any pre-neoplastic disorder (including myelodysplastic syndrome or myeloproliferative neoplasm). In some embodiments, the binding partners described are used prophylactically against any of the types of cancer described.

[0079] In embodiments, administration of one or more binding partners (which may, but need not, be in a pharmaceutical formulation) to an individual in need thereof exhibits improved activity compared to a control. In some embodiments, the control includes a different antibody, a different form of the same antibody / binding partner, or an antibody / binding partner delivered without the addition of an additional agent. In embodiments, the binding partners described herein provide improved antibody dependent cell cytotoxicity (ADCC) or internalization (e.g., for ADC) compared to the control. In embodiments, the control protein or peptide does not include a covalently attached molecule. The control peptide may include the same sequence as the experimental peptide, or, if the experimental peptide includes a mutation, the control peptide may include a wild-type sequence.

[0080] The compositions (e.g., pharmaceutical formulations) of the present disclosure can include only one or more binding partners, and thus include combinations of different binding partners. Similarly, one or more binding partners can be combined with any other therapeutic agent, non-limiting examples of which include traditional chemotherapeutic agents and modulators of T cell costimulatory molecules (often referred to as immune checkpoint inhibitors). T cell costimulatory molecules are known in the art (PMID 30115704) and include, but are not limited to, the following: CTLA4, PD-1, PD-L1, LAG3, TIM3, TIGIT, VISTA, B7-1, B7-2, PD-L2, LSECtin, Galectin-9, CEACAM-1, CD155, CD112, CD28, ICOS, ICOSL, OX40, OX40L, GITR, GITRL, 4-1BB, 4-1BBL, CD40, CD40L, CD27, and CD70. Thus, the present disclosure includes combination therapy using one or more of the described binding partners and any modulator of T cell costimulatory molecules, including but not limited to CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. Non-limiting examples of anti-PD-1 agents include pembrolizumab and nivolumab. Examples of anti-PD-L1 include avelumab and atezolizumab. Examples of anti-CTLA-4 include ipilimumab. The binding partners may be combined with any form of adoptive immunotherapy.

[0081] In embodiments, the disclosure includes administering to an individual in need thereof one or more binding partners and at least one additional agent to provide an additive effect or a more than additive effect, such as a synergistic result. In embodiments, the described effect includes inhibition of cancer growth, inhibition of metastasis, or other beneficial effect. An additive or synergistic effect may also be achieved by using a combination of at least two of the described binding partners.

[0082] A variety of techniques have been developed for the production of binding partners, which are within the scope of the present disclosure. In embodiments, the binding partner is produced in a host cell by a recombinant expression vector. The present disclosure includes all polynucleotide sequences encoding the amino acid sequences described herein, expression vectors comprising such polynucleotide sequences, and in vitro cell cultures comprising such expression vectors. In embodiments, the cell culture comprises prokaryotic or eukaryotic cells. In embodiments, the cell culture is a mammalian cell. In embodiments, the cell is a CHO cell. In embodiments, the cell is a HEK293 cell and derivatives thereof. Kits comprising the binding partner and / or cell cultures expressing the binding partner are provided by the present disclosure. Generally, the kit comprises one or more sealed containers comprising the binding partner or cells expressing the binding partner. Instructions for using the binding partner for therapeutic and / or diagnostic purposes may be included in the kit.

[0083] Cells that may be modified to express any of the described binding partners include CD4 + T cells, CD8 + T cells, natural killer T cells, γδ T cells, and cells that are precursors of T cells, such as hematopoietic stem cells or other lymphoid precursor cells, such as immature thymocytes (double negative CD4 - CD8 - ) cells, or double-positive thymocytes (CD4 + CD8 + ), but are not necessarily limited to. In some embodiments, the progenitor cells include markers such as CD34, CD117 (c-kit), CD90 (Thy-1), and the like. In some embodiments, the modified cells include macrophages. The modified cells described can be used therapeutically or prophylactically.

[0084] In some embodiments, the present disclosure provides for the generation of binding partners. This approach involves providing a plurality of different binding partners, exposing the plurality of different (e.g., separate) binding partners to one or a variety of peptide conjugates, and selecting binding partners that specifically bind to the peptide conjugates that include a covalently conjugated drug or other molecule, but do not bind to proteins or peptides that do not include a covalently conjugated drug or other molecule. As described above, this approach can be performed in a manner that requires or does not require that the amino acid sequence of the protein or peptide be part of an antigenic determinant. The described approach can be used to select binding partners specific for the presentation of peptide conjugates as components of any MHC complex.

[0085] In some embodiments, the binding partners described herein, and those not described but obvious to one of skill in the art, can be used to determine whether a particular drug or other molecule forms a covalent interaction with a protein or peptide. Thus, the present disclosure provides for exposing a protein or peptide substrate to a drug candidate and using a binding partner described herein or identified as described herein to determine whether the drug forms a covalent interaction with the substrate. This determination can be made based on whether the binding partner binds to a protein or peptide covalently attached to the drug. This approach can be used in place of currently available techniques such as mass spectrometry.

[0086] In embodiments, the binding partners of the present disclosure can be used in any immunological diagnostic test, including but not limited to the imaging approaches described above. In embodiments, one or more binding partners described herein can be used as a component of any form of enzyme-linked immunosorbent assay (ELISA), including but not limited to direct ELISA, sandwich ELISA, competitive ELISA, and reverse ELISA. In embodiments, one or more binding partners described herein can also be incorporated into an immunodiagnostic device, such as a microfluidic device, a lateral flow device, etc. The binding partners can also be used, for example, in Western blots and immunoprecipitation assays.

[0087] The following examples are illustrative of the present disclosure and are not intended to be limiting. In some embodiments, the antibody described in Example 3 has different properties than the antibody described in Example 1. Other differences between the binding partners will be apparent from the examples and their associated figures. Different properties include, but are not necessarily limited to, specificity for the drug conjugate displayed in the context of a particular MHC type. Thus, the binding partners may exhibit different binding partners when the peptide conjugate is in a particular MHC complex. Example 1

[0088] In this embodiment, KRAS G12C This invention provides a description of the identification and characterization of a binding partner that specifically binds to ARS-1620, forming a covalent interaction with the . In particular, FIG. 1 demonstrates phage ELISA of phage-displayed antibody clones.

[0089] KRAS G12C -GDP and KRAS G12C The binding of KRAS to the -GDP-ARS-1620 conjugate was measured. G12CFrom these candidates, four distinct antibodies were identified. Of these, 12C-ARS-Fab59 binds KRAS covalently linked to ARS-1620. G12C The results are shown in Figure 2, which shows that KRAS in the GTPγS- or GDP-bound nucleotide state (with or without ARS-1620) was characterized by bead-binding assay (PMID: 33358997). G12C Figure 3 also shows the binding of 12C-ARS Fab59 to KRAS conjugated to ARS-1620. G12C In particular, we have demonstrated that KRAS specifically binds to GTPγS- or GDP-bound nucleotides (with or without ARS-1620 conjugation). G12C 12C-ARS-Fab binding to (left) or WT RAS isoforms (right) is shown.

[0090] Figure 4 shows that ARS-1620 / KRAS was detected by pull-down assay of lysates prepared from cell lines. G12C The use of 12C-ARS-Fab59 to measure adducts was demonstrated. To obtain the data shown in Figure 4, Fab59 was used to measure the adducts of KCP (Kras) and RAS-less MEFs (4A) reconstituted with the indicated KRAS mutants, treated with or without ARS-1620. G12C ; Tp53 R172H Immunoblotting was performed on whole cell lysates and 12C-ARS Fab-pulldowns (PD) from H358 and MIAPaCa-2 cells treated as indicated (Figure 4C), which were subjected to SDS-PAGE and immunoblotting (with anti-pan RAS and anti-ERK2 antibodies, the latter as a loading control). Figure 4D shows ARS adduct formation in samples from 4C, quantified by LC / MS-MS assay. ARS-1620 and SHP099 concentrations were 10 μM in all panels.

[0091] Figure 5 shows that 12C-ARS-Fab59 can be used to measure the binding of ARS-1620 to mutant KRAS by pull-down assay using lysates prepared from animal tissues. In particular, Figure 5AB shows the binding of LSL-KRAS after oral administration of ARS-1620 (200 mg / kg / d) alone or in combination with the SHP2 inhibitor SHP099 (75 mg / kg / d) for 3 days. G12C -Tp53 R270H (A) Tumor and LSL-KRAS G12C (B) Anti-pan RAS and anti-ERK2 (loading control) immunoblots of lysates from tumors and 12C-ARS Fab pulldown (PD). The following materials and methods were used to obtain the results described above.

[0092] RAS nucleotide exchange and generation of RAS conjugated with ARS-1620 Purified RAS(1-174) protein (1) containing 6xHIS tag (SEQ ID NO: 13) and AVI tag used in binding experiments and phage display selection was prepared by diluting the stock protein (usually containing 20-100 μM RAS) 25-fold with 20 mM Tris-Cl buffer (pH 7.5) containing 5 mM EDTA, 0.1 mM DTT, and 1 mM (final concentration) nucleotide (GDP or GTPγS). To generate ARS-bound RAS, ARS-1620 (final concentration: 100 μM) was added together with GDP during the nucleotide exchange reaction of RAS. The sample was incubated at 30 °C for 30 min. Then, to quench the nucleotide exchange reaction, MgCl2 was added to a final concentration of 20 mM, and the solution was incubated on ice for at least 5 min before use.

[0093] ARS-combined KRAS G12C Selection of phage-displayed antibody fragments against The general procedure for the development of Fabs against purified protein targets has been described in (2). Four rounds of phage display library selection were performed with biotinylated KRAS(G12C)-GDP+ARS-1620 at 100 nM, 100 nM, 50 nM, and 20 nM in the first, second, third, and fourth rounds, respectively. In the first round, KRAS G12C -GDP+ARS-1620 binding clones were recovered; in the second round, KRAS G12C -GDP+ARS-1620-binding clone (previously KRAS G12C -GDP precleared) and in the third round, KRAS G12C -GDP+ARS-1620 binding clones were collected (previously KRAS G12C -GTP precleared). In the final round, KRAS G12C -GDP+ARS-1620 binding clones were collected (previously KRAS G12C -GDP). Phages captured on the beads were eluted with 100 μl of 0.1 M Gly-HCl (pH 2.1) and immediately neutralized with 35 μl of 1 M Tris-Cl (pH 8). Recovered clones were analyzed by phage ELISA and DNA sequencing as described (2).

[0094] Bead binding assay The general method was previously described (3). 50 μl of M280 streptavidin beads (Thermo Fisher) were incubated with 100 μl of biotinylated 12C-ARS Fab (30 nM or 4 nM). Ligand-free streptavidin on the beads was blocked by adding excess biotin. The beads were washed with supplemented TBST (50 mM Tris pH 7.5, 150 mM NaCl, 20 mM MgCl2, 0.1 mM DTT, 0.05% Tween-20) and dispensed into wells of a 96-well U-bottom plate (Greiner). The beads were then incubated 1:1 with purified RAS protein diluted in supplemented TBS (50 mM Tris pH 7.5, 150 mM NaCl, 20 mM MgCl2, 0.1 mM DTT) at twice the concentration listed in the titration curve for 30 min (room temperature). The beads containing bound Fab and RAS were transferred to wells of a 96-well filter plate (Millipore, MSHVN4550) and washed twice with supplemented TBST before incubation with Neutravidin-Dylight 650 (Thermo Fisher Scientific) for 30 min at 4°C. The beads were washed twice with supplemented TBST and then resuspended in supplemented TBS for flow cytometry using an iQue screener (Sartorius). The median signal intensity in the Dylight 650 channel of the 75th-95th percentile population was taken as the binding signal to the target. K D was calculated by fitting the binding signals to a 1:1 binding model.

[0095] Expression, purification and characterization of recombinant Fab The phage display vector was converted into a Fab expression vector containing a substrate tag for the biotin ligase BirA at the carboxyl terminus of the heavy chain. The Fab was expressed in Escherichia coli strain 55244 (ATCC) and purified by protein G affinity chromatography followed by cation exchange chromatography (see (2)). The purified Fab was biotinylated in vitro with purified BirA. Approximately 2–5 mg of purified Fab was routinely obtained from 1 L of bacterial culture. The purity of the Fab was >90% by SDS-PAGE.

[0096] KRAS G12C -Adduct assay Cells grown in 6-well plates were treated with ARS-1620 and / or SHP099 as indicated in the figures. Cells were lysed by incubation in GTPase lysis buffer (25 mM Tris-Cl pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, and 5% glycerol) supplemented with protease and phosphatase inhibitors for 15 min on ice immediately prior to analysis. After centrifugation at 15,000 g for 15 min, the supernatant was collected and incubated with streptavidin (SA) agarose resin (Thermo Fisher Scientific) for 1 h at 4°C, followed by a brief centrifugation to reduce nonspecific binding to the resin. The precleared lysate was incubated with biotinylated 12C-ARS-Fab bound to SA agarose for 1.5 h at 4°C with rotation. The agarose beads were then washed twice with GTPase lysis buffer, boiled in 1x SDS-PAGE sample buffer, and immunoblotted with pan-RAS antibody (Millipore).

[0097] Immunoblotting Whole cell lysates were prepared in modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 0.1% SDS, without sodium deoxycholate) and supplemented with protease inhibitors (40 μg / ml PMSF, 2 μg / ml antipain, 2 μg / ml pepstatin A, 20 μg / ml leupeptin, and 20 μg / ml aprotinin) and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, and 10 mM sodium pyrophosphate). After centrifugation in a microcentrifuge to remove debris, samples were quantified using the DC Protein Assay Kit (Bio-Rad). Total lysate proteins were separated by standard SDS-PAGE and transferred to 1X transfer buffer and 15% methanol. Membranes were incubated with respective primary and secondary antibodies labeled with IRDye (680nm and 800nm) and visualized using a LICOR instrument. Monoclonal pan-RAS antibody (clone Ab-3; OP40-100UG; 1:1000) was obtained from Millipore, and mouse monoclonal ERK-2 (D2:sc-1647; 1:1000) was purchased from Santa Cruz Biotechnology.

[0098] KRAS G12C LC / MS-MS assay of ARS binding to cells (5×10 5 ) were treated with the indicated compounds for the indicated times, then washed twice with PBS and prepared for protein extraction and LC / MS-MS analysis (see (4)). LC / MS-MS was performed at the PCC Proteomics Shared Resource at the New York University School of Medicine.

[0099] Using similar methods, the results described in Example 2 were obtained. The antibodies described in this example are as follows: Examples of antibody clones that bind to the KRAS(G12C)-ARS-1620 conjugate: CDR residues in bold (Kabat numbering).

[0100] TIFF2024513651000004.tif176169

[0101] List of References Related to Example 1 TIFF2024513651000005.tif148169 Example 2

[0102] This example describes a binding partner that specifically binds AMG510 covalently attached to a peptide. To obtain the results described in this example, several methods described in Example 1 were applied. In this example, AMG510 (purchased from Selleckchem) was conjugated to: A peptide corresponding to KRAS(G12C) residues 4-18 H2N-YKLVVVGACGVGKSA(dPEG4)(K-long chain biotin)-amide (SEQ ID NO:22) Poly-Ser peptides with a central Cys: H2N-SSSSCSSSSW(K-long chain biotin)-amide (SEQ ID NO:23)

[0103] Using these peptides as targets, established methods (1-3) We screened a human single-chain Fv yeast display library by the method described above. After multiple rounds of library selection, individual clones were screened. We developed three antibodies that bound to AMG510 conjugated to both KRAS(G12C) and poly-Ser peptide (Figure 6). As a result, these antibodies mainly recognized the AMG510 moiety and did not recognize the peptide portion of the conjugate. In addition, we developed other clones that were selective for AMG510 conjugated to KRAS(G12C) peptide.

[0104] One such clone, P2AMR-1, was produced in a human IgG1 format and further characterized. This clone bound AMG510 conjugated to the KRAS(G12C) peptide with high apparent affinity in bead-binding assays (Figure 7) (4). This antibody clone also bound tightly to AMG510 conjugated to the shorter KRAS(G12C) peptide, VVGACGVGK (SEQ ID NO: 1), in the context of HLA-A*03:01 (BioLegend Flex-T) (Figure 8).

[0105] P2AMR-1 detected AMG510 conjugated to KRAS(G12C) peptide added to Raji cells, known to express HLA-A*03:01. In contrast, P2AMR-1 did not detect KRAS(wild type) peptide loaded in a similar manner (Figure 16). Furthermore, P2AMR-1 did not bind to AMG510 conjugated to KRAS(G12C) peptide added to cells not known to express HLA-A*03:01, such as MV4-11 cells and Expi293 cells (Figure 16).

[0106] These results indicate that the antibodies provided in the present disclosure (binding partners of the present disclosure) recognize the AMG510 moiety in a conjugation partner-independent manner and suggest that our antibodies and their derivatives can be used to identify cells that present AMG510-KRAS(G12C) peptide conjugates on MHC molecules on the cell surface.

[0107] More generally, these results suggest a method for targeting any cell that possesses an intracellular target that forms a covalent adduct with a small molecule ligand.

[0108] This example is limited to AMG510 covalent modification of the above substrate and shows the following non-limiting binding partners: CDR residues (Kabat scheme) are shown in bold.

[0109] TIFF2024513651000006.tif112169

[0110] List of References Related to Example 2 TIFF2024513651000007.tif119169 Example 3

[0111] In this example, we describe an antibody that binds to a peptide-drug conjugate (only in the context of specific MHC display of said peptide-drug conjugate). The antibody was generated as follows.

[0112] Antigen preparation KRAS(G12C) peptides (H2N-VVGACGVGK-OH (SEQ ID NO: 1) and H2N-VVVGACGVGK-OH (SEQ ID NO: 2)) were reacted with AMG510 (Selleckchem) and loaded onto Flex-T HLA-A*03:01 and Flex-T HLA-A*11:01 (Biolegend) or onto HLA-A*03:01 and HLA-A*11:01 produced in-house. KRAS(WT) peptide (H2N-VVGAGGVGK-OH (SEQ ID NO: 29)) was similarly loaded onto HLA molecules. EGFR peptide (H2N-QLMPFGCLL-OH (SEQ ID NO: 30)) was reacted with osimertinib (Selleckchem) and loaded onto Flex-T HLA-A*02:01 or onto HLA-A*02:01 produced in-house. As a control, the same peptide was reacted with beta-mercaptoethanol and loaded onto the HLA molecule. BTK peptide (H2N-YMANGCLLNY-OH (SEQ ID NO: 31)) was reacted with ibrutinib (Selleckchem) and loaded onto Flex-T HLA-A*01:01 or HLA-A*01:01 produced in-house. As a control, the same peptide was reacted with beta-mercaptoethanol and loaded onto the HLA molecule. The peptide-loaded HLA mixture prepared with Flex-T HLA protein was used without further purification. The peptide-loaded HLA mixture prepared with the HLA sample produced in-house was further purified by size exclusion chromatography using a Superdex S200 column.

[0113] Sorting of antibody phage-display libraries Sorting of the antibody phage display library was performed as previously reported. (1)Briefly, the phage-display library was first sorted with all four antigens (100 nM) in the first round, followed by sorting with a single antigen at 100, 50, and 20 nM in the second, third, and fourth rounds, respectively. To enrich for clones with the desired specificity, counterselections were performed with KRAS(WT) peptide-loaded MHC molecules or beta-mercaptoethanol-treated peptide-loaded MHC molecules in the second, third, and fourth rounds. Binding of individual phage clones was determined using a multiplex bead-binding assay (2) was used for testing.

[0114] Sorting of antibody yeast-display libraries and characterization of clones Display of antibody clones in the form of single-chain Fv (scFv) on the yeast surface, library sorting using fluorescence-activated cell sorting, and characterization of individual clones were performed essentially as previously described (Hattori et al. PMID 23955773; Cao et al. PMID 17406305).

[0115] Deep Mutational Scanning Deep mutational scanning was performed according to the general procedure previously published (PMID 32841599). The yeast-display library (scFv format) contained mutants diversified at one position at the NNK codon. The yeast-display library was subjected to FACS with the antigen of interest to enrich for a pool of antigen-binding and non-antigen-binding clones. The DNA sequences of the enriched pools were determined and amino acid substitutions were inferred.

[0116] Antibody production The genes encoding the selected antibody clones were transferred from the phage display vector to IgG expression vectors (pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hK: InvivoGen), and IgG protein was produced using the ExpiCHO cell line (Thermo Fisher) and purified by Protein Capture Devices (GORE) using Protein A. The data presented in this example, in conjunction with Figures 9-15 and 17, provides the following information:

[0117] Figure 9 is a schematic representation of the disclosed concept called HapImmune. Numbers 1-7 indicate the relevant steps. 1. A covalent inhibitor is administered and enters cells that harbor the target protein. 2. The inhibitor binds to the target and forms a covalent bond with the target. 3 and 4. As part of natural protein turnover (or induced proteolysis in the case of PROTACs), the target-drug conjugate is degraded by the proteosome system, resulting in the production of a drug-conjugated peptide. 5. The peptide conjugate is incorporated into a compatible MHC molecule. 6. The MHC / peptide-drug conjugate complex is transported to the cell surface. The HapImmune antibody recognizes this complex. 7. Surface-bound antibodies recruit immune effector cells, such as NK cells, resulting in cell-killing activity. Multiple modalities are envisioned to effect cell-killing activity, including ADCC, ADCP, CDC, BiTE, CAR-T, CAR-NK, ADC, and radioisotope conjugates, but are not explicitly depicted here.

[0118] FIG. 10 shows development data for antibodies that bind to MHC / peptide-drug conjugate complexes. (A) Multiplex bead-binding assay (MBBA) of phages displaying different antibody clones (1). Each phage clone was tested for binding to a total of five antigens displayed on the beads: a complex of HLA-A*03:01 with KRAS(G12C) peptide conjugated to AMG510 (denoted as HLA-A*03:01_RAS-AMG510 in the figure); a complex of HLA-A*03:01 with KRAS(wild type) peptide (denoted as HLA-A*03:01_WTRAS); a complex of HLA-A*11:01 with KRAS(G12C) peptide conjugated to AMG510 (denoted as HLA-A*11:01_RAS-AMG510); a complex of HLA-A*11:01 with KRAS(wild type) peptide (denoted as HLA-A*11:01_WTRAS); and beads displaying no antigen (denoted as no target). (B) MBBA assay of phage displaying different antibody clones on: HLA-A*01:01 in complex with BTK peptide conjugated to ibrutinib (denoted as HLA-A*01:01_BTK-ibrutinib in the figure); HLA-A*01:01 in complex with BTK peptide conjugated to beta-mercaptoethanol (denoted as HLA-A*01:01_BTK-βme); and beads displaying no antigen (denoted as no target). (C) MBBA assay of phages displaying different antibody clones on: HLA-A*02:01 in complex with EGFR peptide conjugated to osimertinib (denoted as HLA-A*02:01_EGFR-osimertinib in the figure); HLA-A*02:01 in complex with EGFR peptide conjugated to beta-mercaptoethanol (denoted as HLA-A*02:01_EGFR-βme); and beads not presenting antigen (denoted as no target).

[0119] Figure 11 shows the results of a binding titration using a multiplex bead-binding assay (MBBA) of purified antibodies targeting the KRAS(G12C)-AMG510 conjugate. The clone names are shown above each graph. Antigen nomenclature is described in Figure 10. The left column shows the binding data to the HLA-A*03:01 complex and the right column shows the binding data to the HLA-A*11:01 complex. The apparent dissociation constants (K D ) values ​​were determined using nonlinear least-squares fitting of the 1:1 binding function. The wild-type RAS peptide complex and no target data were all close to baseline and overlapped, so the apparent K D Values ​​were not determined. Data presented here are from triplicate determinations. Error bars are within the symbol sizes.

[0120] Figure 12 shows that antibody binding to drug-peptide conjugates complexed to MHC is not affected by the presence of free drug. MBBA binding signals of selected "AMR" series antibodies to HLA-A*03:01 complexed with KRAS(G12C) peptide conjugated to AMG510 are shown in the absence (white bars) and presence (grey bars) of 10 μM free AMG510. Antibody concentrations were adjusted to obtain subsaturating signals and are shown in brackets. Data shown are from triplicate determinations.

[0121] Figure 13 shows the results of a binding titration using a multiplex bead-binding assay (MBBA) of purified antibodies targeting the BTK-ibrutinib conjugate. The clone names are listed above each graph. The antigen nomenclature is described in Figure 10. The apparent dissociation constants (K D ) values ​​were determined using nonlinear least-squares fitting of the 1:1 binding function. The beta-mercaptoethanol-binding peptide complexed with HLA-A*01:01 and no target data were all close to baseline and overlapped, so the apparent K D Values ​​were not determined. Data presented here are from triplicate determinations. Error bars are within the symbol sizes.

[0122] Figure 14 shows the results of a binding titration of purified antibodies to KRAS(G12C)-AMG510 conjugates presented by endogenous MHC molecules on the cell surface. Raji cells were first incubated with KRAS(G12C)-AMG510 conjugates or KRAS (wild type) peptide, and excess conjugate and peptide were washed away. The amount of surface-bound antibody detected with a fluorescently labeled secondary antibody is shown as a function of the IgG concentration used for staining. The apparent dissociation constant (K D ) values ​​were determined using nonlinear least-squares fitting of the 1:1 binding function. Data shown are from triplicate determinations.

[0123] Figure 15 shows the results of antibody binding to KRAS(G12C)-expressing cell lines pretreated with AMG510. The non-small cell lung cancer cell line H358 was incubated with AMG510 for 2 days and stained with an antibody targeting the KRAS(G12C) peptide-AMG510 conjugate or an isotype control, followed by detection with a secondary antibody. (A) Flow cytometry histogram (B) Quantification of median fluorescence intensity in H358 cells treated with and without AMG510. Antibodies used are indicated on the horizontal axis. (C) Quantification of median fluorescence intensity of H358 cells and HEK293T cells (negative control) treated with or without AMG510 and stained with AMRA3-7 antibody.

[0124] Figure 16 relates to Example 2 and shows the binding of P2AMR-1 IgG to cells preincubated with KRAS(G12C) peptide-AMG510 conjugate, KRAS(wild type) peptide, or no peptide. The antibody was precomplexed with a dye-labeled secondary antibody to increase effective binding (avidity). When Raji cells expressing HLA-A*03:01 were incubated with the conjugate, the antibody bound to the Raji cells.

[0125] FIG. 17 shows binding of purified antibodies in IgG format to the indicated drug-peptide / MHC complexes as measured using a multiplex bead binding assay (MBBA).

[0126] 18 and 19 will be described in the fourth embodiment.

[0127] 20 shows the results of binding titration curves of yeast surface-displayed AMR-A3-7 and AMR-A3-7D to HLA-A*03:01 presenting AMG510 conjugated to Cys residues of the 9-mer and 10-mer RAS(G12C) peptides, VVGACGVGK (SEQ ID NO: 1) and VVVGACGVGK (SEQ ID NO: 2), respectively.

[0128] Figures 23 and 24 show the results of deep mutational scanning of the CDR-L3 and CDR-H3 of AMR-A3-7D and OEA2-5, respectively.

[0129] Specific, non-limiting examples of antibody sequences that specifically bind to MHC-drug conjugates include the following: Examples of antibody clones that bind to the KRAS(G12C)-AMG510 conjugate presented by HLA-A*03:01 and HLA-A*11:01. CDR residues in bold (Kabat scheme).

[0130] TIFF2024513651000008.tif223169

[0131] TIFF2024513651000009.tif223169

[0132] TIFF2024513651000010.tif224169

[0133] TIFF2024513651000011.tif225169

[0134] TIFF2024513651000012.tif179169

[0135] Antibody clones that preferentially bind to the KRAS(G12C)-AMG510 conjugate presented on HLA-A*03:01 relative to the KRAS(G12C)-AMG510 conjugate presented on HLA-A*11:01 CDR residues are in bold (Kabat scheme).

[0136] TIFF2024513651000013.tif180169

[0137] TIFF2024513651000014.tif135169

[0138] An antibody clone that preferentially binds to the KRAS(G12C)-AMG510 conjugate presented on HLA-A*11:01 relative to the KRAS(G12C)-AMG510 conjugate presented on HLA-A*03:01.

[0139] TIFF2024513651000015.tif225169

[0140] TIFF2024513651000016.tif225169

[0141] TIFF2024513651000017.tif180169

[0142] Example of an antibody clone that binds to the BTK-ibrutinib conjugate presented on HLA-A*01:01. CDR residues are in bold (Kabat scheme).

[0143] TIFF2024513651000018.tif224169

[0144] TIFF2024513651000019.tif89169

[0145] Example of an antibody clone that binds to EGFR-osimertinib conjugate presented by HLA-A*02:01. CDR residues are in bold (Kabat scheme).

[0146] TIFF2024513651000020.tif226169

[0147] TIFF2024513651000021.tif45169

[0148] References related to Example 3: TIFF2024513651000022.tif60169 Example 4

[0149] This example demonstrates the single chain bispecific (scDb) format of the Hapimmune antibody and its cell killing efficacy. Data from non-limiting embodiments are shown in Figures 18 and 19. Results are summarized in the brief descriptions of Figures 18 and 19.

[0150] To obtain the results in Figure 18, Raji and T2 cells (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. The cytotoxic effect of scDbs was measured according to a previously published protocol (ref: PMID 26813960). Briefly, Raji or T2 cells were stained with carboxyfluorescein succinimidyl ester (CSFE, ThermoFisher, 65-0850-84) and incubated with KRAS(G12C)-AMG510 conjugates at a final concentration of 10 μM or EGFR-osimertinib at 1 μM in the presence of 10 μg / mL human β2 microglobulin for 4 h. Cells were harvested by centrifugation and washed with medium to remove unbound conjugates and peptides. Next, the peptide-drug pulsed cells were co-cultured with human T cells (E:T=3:1) in the presence of single-chain bispecific antibodies (scDb) for 19-21 hours. After incubation, the cells were harvested, washed with PBS, and stained with Fixable Viability Dye eFluor660 (ThermoFisher, 65-0864-14). After washing the cells, they were analyzed with an iQue screener (Sartorius).

[0151] For the results in Figure 19, lung cancer cell lines were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. 4 Cells / well were seeded in 96-well flat-bottom plates and incubated at 37°C, 5% CO2 for 24 hours. The medium was replaced with fresh medium supplemented with 100 nM AMG510 or DMSO, and cells were incubated at 37°C for 24 hours. After incubation, cells were co-cultured with human T cells (E:T=5:1) and AMRA3-7_UCHT1 scDb in the presence of 100 IU / mL IL-2 for 24 hours at 37°C. Cell viability was assessed using PrestoBlue TM Cell Viability Reagent (ThermoFisher, A13261) was used to assess cytotoxicity. Cytotoxicity was calculated by subtracting the fluorescence signal of wells containing only T cells from the fluorescence signal of a well and normalizing it to the fluorescence signal of wells without scDb.

[0152] An example of a single-chain bispecific antibody clone targeting both HLA-A*03:01_RAS-AMG510 and HLA-A*11:01_RAS-AMG510 The italicized sequences represent the AviTag and HisTag, respectively. TIFF2024513651000023.tif132169

[0153] Example of a single-chain bispecific antibody clone targeting HLA-A*02:01_EGFR-osimertinib. The italicized sequences represent the AviTag and HisTag, respectively. TIFF2024513651000024.tif128169 Example 5

[0154] This example demonstrates scDb and 2+1 CrossMab antibodies constructed using the AMRA3-7 clone and their efficacy in cell killing. Data from non-limiting embodiments are shown in Figures 21 and 22.

[0155] To obtain the results in Figure 21(A) and Figure 22(B), Raji cells (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. The cytotoxicity of scDbs and CrossMabs was measured according to a previously published protocol (ref: PMID 26813960). Briefly, Raji cells were stained with carboxyfluorescein succinimidyl ester (CSFE, ThermoFisher, 65-0850-84) and incubated with 10 μM KRAS(G12C)-AMG510 conjugate or 10 μM KRAS(WT) peptide (final concentration) in the presence of 10 μg / mL human β2 microglobulin for 4 h. Cells were harvested by centrifugation and washed with medium to remove unbound conjugate and peptide. The peptide-drug pulsed cells were co-cultured with human T cells (E:T=3:1) in the presence of scDb or CrossMab for 19 hours. After incubation, the cells were harvested, washed with PBS, and stained with Fixable Viability Dye eFluor660 (ThermoFisher, 65-0864-14). After washing again, the cells were analyzed using an iQue screener (Satorius).

[0156] To obtain the results in Figure 21(B) and Figure 22(C), H2122 cells (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. In the cytotoxicity assay, 5 × 10 3Cells / well were seeded into 96-well flat-bottom plates and incubated for 48 h at 37°C, 5% CO2. After incubation, cells were co-cultured with human T cells (E:T=10:1) and AMRA3-7D scDb or CrossMab in the presence of 10 ng / mL IL7 and IL15 for 24 h at 37°C. Cell death was measured using the CytoTox-Glo cytotoxicity assay (Promega, G9290). The luminescence signal of a given well was calculated by subtracting the signal from wells containing H2122 and T cells but no scDb or CrossMab constructs.

[0157] To obtain the results in Figure 22(A), Jurkat and Raji cells (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. The cells were washed twice with PBS and then incubated with AMRA3-7D CrossMab for 30 min at 4°C. After washing three times with PBS containing 1% BSA (PBS / BSA), the cells were stained with Alexa647 Goat Anti-Human IgG Fc (Jackson ImmunoResearch, 109-605-098). After incubation, the cells were washed three times with PBS / BSA and analyzed on an iQue screener (Sartorius).

[0158] TIFF2024513651000025.tif62169

[0159] TIFF2024513651000026.tif209169

[0160] FIG. 21 shows the cytotoxic effect of AMRA3-7D in a single-chain bispecific antibody (scDb) format. (A) Raji cells were first pulsed with AMG510 conjugated to a peptide corresponding to the KRAS(G12C) fragment or a control peptide corresponding to KRAS(wild type). The pulsed cells were co-cultured with human T cells (effector:target=3:1) in the presence of the indicated concentrations of scDb. After 17 h of culture, dead cells were stained and detected by flow cytometry. (B) H2122 cells were first incubated with AMG510 or DMSO. They were then co-cultured with human T cells (effector:target = 10:1) in the presence of the indicated concentrations of scDb. After incubation, dead cells were measured by detecting distinct intracellular protease activity released from membrane-compromised cells. Data shown here are from triplicate determinations. Error bars indicate sd. When error bars are not visible, the error is smaller than the symbol. Anti-HLA-A3 is the positive control.

[0161] FIG. 22 shows the CD3 binding properties and cytotoxic effect of AMRA3-7D in CrossMab format. (A) Binding titration curves of AMRA3-7 CrossMab to Jurkat (CD3 positive) and Raji (CD3 negative) cells. Apparent K D Values ​​are shown. Cells were not pulsed with peptide. (B, C) Cytotoxic effect of AMRA3-7D CrossMab on Raji cells pulsed with exogenous peptide-drug conjugates (B) and on H2122 cells treated with the drugs (C). Methods are the same as in Fig. 22. Data shown here are from triplicate determinations. Error bars indicate sd. When error bars are not visible, the error is smaller than the symbol. Anti-HLA-A3 serves as a positive control. Example 6

[0162] This example shows deep mutational analysis of the AMR-A3-7D and OEA2-5 antibodies. Data from non-limiting embodiments are shown in Figures 23 and 24. To identify mutations in the CDRs of AMR-A3-7D and OEA2-5 that retain antigen binding, we performed deep mutational scanning of CDR-L3 and CDR-H3 residues. In the yeast display format, each residue in CDR-L3 and CDR-H3 was mutated one residue at a time to all genetically encoded amino acids using the NNK codon (N=A, T, G, and C; K=G and T). The resulting mutant pools were combined and the libraries were subjected to FASC with the relevant antigens, i.e., AMG510-KRAS(G12C) peptide complexed to HLA-A*03:01 for AMR-A3-7D and osimertinib-EGFR complexed to HLA-A*02:01 for AMR-A3-7D. Different antigen concentrations were used to adjust the stringency of library selection. Vectors recovered from the binding and non-binding pools were deep sequenced on an Illumina MiSeq instrument. Mutations found in the different pools were inferred from DNA sequencing analysis.

[0163] From this analysis, the present disclosure shows the following allowed mutations at each CDR position, as shown in the table below. For reference, the VL and VH sequences of the parent clones are shown, with the analyzed CDR residues in bold italics. In embodiments, the present disclosure includes each mutation alone, and all combinations of mutations. Thus, as is evident from the table, the present disclosure includes the described CDRs with 1, 2, 3, 4, 5, 6, 7, or 8 mutations as shown in the table. The present disclosure also includes the possibility of additional amino acids (e.g., in CDR1, CDR2, and in framework sequences).

[0164] TIFF2024513651000027.tif60169

[0165] TIFF2024513651000028.tif110169

[0166] TIFF2024513651000029.tif91169

[0167] Deep mutational scanning of OEA2-5 TIFF2024513651000030.tif53169

[0168] TIFF2024513651000031.tif82169

[0169] TIFF2024513651000032.tif89169

[0170] As can be seen from the above table, in one embodiment the binding partner comprises a light chain comprising a complementarity determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 154 and a heavy chain comprising a CDR3 comprising the sequence of SEQ ID NO: 156. In another embodiment the binding partner comprises a light chain comprising a CDR3 comprising the sequence of SEQ ID NO: 158 and a heavy chain comprising a CDR3 comprising the sequence of SEQ ID NO: 160.

[0171] FIG. 23 shows deep mutational scanning analysis of CDR-L3 and H3 residues of AMR-A3-7D. (a) Representative flow cytometry profiles of yeast cells displaying AMRA3-7D or its deep mutational scanning library populations, measuring binding to 5 nM HLA-A*03:01 presenting AMG510 conjugated to Cys residues in the peptide VVGACGVGK (SEQ ID NO:1). The profile of the parent antibody, AMRA3-7D, is shown on the left, and profiles of sorted subsets of the deep mutational scanning library are shown on the right. Libraries were sorted using 1, 3, and 10 nM of target (referred to as conditions 1, 2, and 3, respectively), and the non-binder pool was sorted using 50 nM of target. (b) Mutation rates in sorted subsets of deep mutational scanning libraries are shown in heatmap format. The number of deep sequencing reads was normalized to the total number of reads in each sorted pool and multiplied by 1000. Crosses indicate wild-type amino acids.

[0172] FIG. 24 shows the results of deep mutational scanning analysis of the CDR-L3 and H3 residues of OEA2-5. (a) Representative flow cytometry profiles of yeast cells displaying OEA2-5 in single-chain Fv format and the deep mutational scanning library population. Binding was measured to 1.5 nM streptavidin tetramer saturated with biotinylated HLA-A*02:01 presenting osimertinib conjugated to Cys residues of peptide QLMPFGCLL (SEQ ID NO: 30). Profiles of the parent antibody OEA2-5 are shown on the left and profiles of sorted subsets of the deep mutational scanning library are shown on the right. Libraries were sorted using 12.5, 2.5, and 0.5 nM of target (referred to as conditions 1, 2, and 3, respectively) and the non-binder pool was sorted using 12.5 nM of target. (b) Mutation rates in sorted subsets of deep mutational scanning libraries are shown in heatmap format. The number of deep sequencing reads was normalized to the total number of reads in each sorted pool and multiplied by 1000. Crosses indicate wild-type amino acids.

[0173] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

Claims

1. A binding partner that specifically binds to a site on a peptide conjugate comprising a peptide and a covalently attached molecule, and that specifically binds to the peptide conjugate in a complex with a major histocompatibility complex (MHC).

2. 2. The binding partner of claim 1, wherein the binding partner does not bind or binds with a lower affinity to a control peptide that does not contain a covalently attached molecule.

3. The binding partner may be an intact antibody, an antigen-binding (Fab) fragment, a Fab' fragment, or a (Fab') 2 3. The binding partner of claim 1 or 2, which is comprised in a fragment, Fd, Fv, dAb, single domain fragment or single monomeric variable antibody domain, single chain bispecific antibody (single chain diabody: scDb), single chain variable fragment (scFv), bispecific T cell engager (BiTE), bispecific killer cell engager (BiKE), CrossMab, trispecific binding partner, or chimeric antigen receptor (CAR).

4. 3. The binding partner of claim 1 or 2, wherein the binding partner is a BiTE comprising (i) a first antigen-binding domain that binds to a peptide conjugate complexed with MHC and a second antigen-binding domain that binds to CD3 on a T cell, (ii) comprises a radioconjugate, (iii) is conjugated to a drug to form an antibody-drug conjugate (ADC), or (iv) comprises a constant region.

5. The binding partner of claim 4, wherein the constant region is an Fc region.

6. The binding partner of claim 5 , wherein the constant region-containing binding partner is adapted to exert cell-killing activity.

7. The binding partner of claim 6 , wherein the cell-killing activity comprises antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or any combination thereof.

8. The binding partner of any one of claims 1 to 7, wherein the peptide is comprised in a segment of a protein associated with cancer.

9. The binding partner of any one of claims 1 to 8, wherein the peptide comprises a mutation.

10. 10. The binding partner of any one of claims 1 to 9, wherein the peptide conjugate is comprised in a protein having enzymatic activity and the covalently attached molecule is an inhibitor of said protein having enzymatic activity.

11. The peptide is a RAS protein selected from the group consisting of KRAS, HRAS, and NRAS; an epidermal growth factor receptor (EGFR) family member selected from Bruton's tyrosine kinase (BTK), EGFR (ERBB1), HER2 / NEU (ERBB2), HER3 (ERBB3), and HER4 (ERBB4); MET (HGFR); fibroblast growth factor receptor (FGFR); cyclin-dependent kinase (CDK); acetylcholinesterase. The binding partner of any one of claims 1 to 9, which is derived from: (ACHE); p90 ribosomal S6 kinase (RSK); TP53; IDH1; GNAS; FBXW7; CTNNB1; DNMT3A; a cathepsin selected from the group consisting of cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W and cathepsin X; a caspase; pancreatic lipase; or METAP2.

12. The peptide is derived from KRAS and the peptide conjugate is G12C The binding partner of claim 11, wherein the covalently linked molecule containing the mutation comprises a G12C inhibitor.

13. The binding partner of claim 11, wherein the G12C inhibitor is selected from the group consisting of AMG510, 2E07, 6H05, SML-8-73-1, MRTX849, JNJ74699157, LY3499446, ARS-853, ARS-1620, MRTX1275, and derivatives thereof.

14. Binding partner according to claim 12 or 13, wherein the peptide is derived from KRAS and comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and the covalently bound inhibitor is AMG510.

15. A binding partner according to any one of claims 12 to 14, wherein the MHC is HLA-A*03:01 or HLA-A*11:

01.

16. 16. The binding partner according to any one of claims 1 to 15, wherein the binding partner comprises a light chain variable region (VL) comprising a light chain (LC) complementarity determining region (CDR) 3 having the amino acid sequence set forth in SEQ ID NO: 154, and a heavy chain variable region (VH) comprising a heavy chain (HC) CDR3 having the amino acid sequence set forth in SEQ ID NO:

156.

17. The binding partner is (i) HC CDR1 of the amino acid sequence DYSIH, HC CDR2 of the amino acid sequence SISSSSGSTSYADSVKG, and 1 X 2 X 3 X 4 X 5 X 6 VH with HC CDR3 of MDY, where X 1 is G and X 2 is selected from the group consisting of A, C, E, G, H, K, L, M, N, P, Q, R, S, T, W and Y; X 3 is selected from the group consisting of G, P, R, T and W; 4 is selected from the group consisting of A, D, E, F, G, H, I, K, M, N, Q, R, S, T, W and Y; X 5 is selected from the group consisting of A, P and V; X 6 is selected from the group consisting of A, C, G, K, L, M, Q, R, S, T and Y, where X 1 X 2 X 3 X 4 X 5 X 6 are mutated relative to the parent sequence of SEQ ID NO: 155; and / or (ii) an LC CDR1 having the amino acid sequence RASQSVSSAVA, an LC CDR2 having the amino acid sequence SASSLYS, and an LC CDR3 having the amino acid sequence QQX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 VL having a LC CDR3 of T, where X 1 is selected from the group consisting of A, I, L, P, S, T and V; 2 is selected from the group consisting of K, R, S and T; X 3 is F or Y, and X 4 is selected from the group consisting of I, R, T and V; 5 is selected from the group consisting of A, E, H, K, L, M, N, Q, R, S, T and Y; X 6 is K or R, and X 7 is selected from the group consisting of A, C, D, E, G, H, K, L, M, N, P, Q, R, S, T, V and W; X 8 is selected from the group consisting of I, L and V, 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 are mutated relative to the parent sequence of SEQ ID NO:

153. The binding partner of any one of claims 1 to 16, comprising:

18. The binding partner is (i) a VH having an HC CDR1 of the amino acid sequence DYSIH, an HC CDR2 of the amino acid sequence SISSSSGSTSYADSVKG, and an HC CDR3 of the amino acid sequence GGWYPAMDY; and (ii) a VL having an LC CDR1 of the amino acid sequence RASQSVSSAVA, an LC CDR2 of the amino acid sequence SASSLYS, and an LC CDR3 of the amino acid sequence QQISYVKKLIT; The binding partner of any one of claims 1 to 17, comprising:

19. A binding partner according to any one of claims 1 to 18, wherein the binding partner comprises a VH having at least 90% sequence identity to SEQ ID NO:4 and a VL having at least 90% sequence identity to SEQ ID NO:

3.

20. A pharmaceutical composition for use in the treatment of cancer, comprising a binding partner according to any one of claims 1 to 19.

21. 21. The pharmaceutical composition of claim 20, wherein the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma or other brain cancer, gastric cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or other skin cancer, sarcoma, or blood cancer.

Images