MANABODY TARGETING P53 TUMOR ANTIGEN AND METHODS OF USE

Abstract

Described herein are methods and compositions for evaluating a mammal having or suspected of having cancer and / or for treating a mammal having cancer. For example, molecules that include one or more antigen binding domains (e.g., single chain variable fragments (scFvs)) capable of binding to a modified peptide (e.g., a tumor antigen) and methods for using such molecules are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 290,353, filed December 16, 2021, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled "44807-0407WO1_SL_ST26.XML". The XML file was created on December 8, 2022 and is 82,067 bytes in size. The contents within the XML file are incorporated herein by reference in their entirety.

[0003] Federally Sponsored Research or Development This invention was made with Government support under Grant CA006973 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0004] Technical Field Described herein are methods and compositions for evaluating a mammal having or suspected of having cancer and / or for treating a mammal having cancer. For example, molecules that include one or more antigen binding domains (e.g., single chain variable fragments (scFvs)) capable of binding to a modified peptide (e.g., a tumor antigen) and methods for using such molecules are provided. [Background technology]

[0005] background In recent years, antibody and cell-based immunotherapies have emerged as promising cancer treatments. However, many immunotherapies that target antigens upregulated in tumor cells have significant toxicity due to the low expression of the same targets in normal cells. An ideal immunotherapy would be able to specifically identify cancer cells that harbor key driver mutations while leaving normal cells alone. Targeting cells that contain these mutations is often difficult because there are many common mutations in driver genes that code for intracellular proteins that are not directly accessible to immunotherapy.

[0006] Somatic mutations in cancer are ideal targets for cancer therapy because they are expressed only in tumor cells and not in normal cells. Targeting driver proteins (broadly subdivided into oncogene proteins and tumor suppressor proteins) has additional benefits. First, these mutations typically occur early in tumor development, so essentially all daughter cancer cells contain the mutation. Second, resistance is less likely to occur, since tumors rely on their ability to confer oncogenicity. Finally, driver proteins tend to have hotspot mutations shared among many patients, so therapies targeting single mutations can be applied to a broad patient population.

[0007] Most mutant proteins, including most mutant driver gene proteins, are intracellular. Although small molecules can target intracellular proteins, the development of small molecules that can specifically inhibit the activity of mutant driver genes, but not their wild-type (WT) counterparts, has remained elusive for the majority of such driver gene proteins. Antibodies that can have the ability to discriminate single amino acid mutations can typically only target extracellular epitopes.

[0008] The immune system samples the intracellular contents of cells through the processing and presentation of antigens. Following proteolysis of proteins, the resulting peptide fragments are loaded onto human leukocyte antigens (HLA) and delivered to the cell surface where T cells function to discriminate between self and non-self peptides via their T cell receptors (TCRs). For example, a virus-infected cell will present viral peptides to its HLA, triggering the T cell to kill the cell. Similarly, in cancer, mutant peptides can be presented to HLA on the cancer cell surface, termed MANA, short for Mutation-Associated Neo-Antigens. In some cases, patients can mount an anti-cancer T cell response to these mutant peptide-HLA neoantigens, to varying degrees, and checkpoint blockade antibodies can further enhance this response. However, many patients, especially those with low mutational loads, are unable to mount an adequate anti-cancer T cell response. Thus, therapeutics or diagnostics that specifically target MANA can provide a truly tumor-specific method for diagnosing or treating cancer.

[0009] HLA class I proteins are present in all nucleated cells. There are three classical HLA class I genes, A, B, and C, each of which is highly polymorphic. Each HLA allele has a specific peptide-binding motif, such that only certain peptides bind to a particular HLA allele.

[0010] There is a continuing need in the art to develop new and improved methods for diagnosing, monitoring, and effectively treating cancer. Summary of the Invention

[0011] overview Identifying therapeutic targets that are highly specific to cancer cells remains one of the greatest challenges for developing effective cancer treatments.

[0012] Described herein are methods and compositions for treating a mammal with cancer. For example, this document provides methods and materials for using one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified peptide (e.g., a modified peptide present in a peptide-HLA-β-2 microglobulin (b2M or β2M) complex) to treat a mammal with cancer (e.g., a cancer expressing a modified peptide). In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified peptide (e.g., a modified peptide present in a peptide-HLA-β2M complex) can be administered to a mammal with cancer (e.g., a cancer expressing a modified peptide) to treat the mammal.

[0013] As shown herein, scFvs have been identified that target (e.g., bind) multiple MANAs present in HLA-restricted MANAs derived from the common cancer driver mutation p53 R175H ("R175H"). Also, as shown herein, scFvs have been used to design bispecific antibodies that can recognize and kill cells expressing MANA (e.g., cancer cells) and induce MANA-dependent T cell activation.

[0014] MANA can be used as a highly specific cancer target because it is not present in normal tissue(s). The ability to specifically target MANA provides a tumor-specific method for diagnosing and / or treating cancer. For example, scFvs that specifically target MANA can be used in full-length antibodies or fragments thereof, antibody-drug conjugates (ADCs), antibody-radionuclide conjugates, T cells expressing chimeric antigen receptors (CARTs), or bispecific antibodies to diagnose and / or treat mammals with cancer. Furthermore, antibodies capable of binding to MANA (MANAbodies), or fragments thereof capable of binding to MANA, have the potential to become widely applicable, genetically predictable, readily available targeted cancer immunotherapy.

[0015] In recent years, antibody and cell-based immunotherapies have emerged as promising cancer treatments. However, many immunotherapies that target antigens upregulated in tumor cells have significant toxicity due to the low expression of the same targets in normal cells. An ideal immunotherapy would be able to specifically identify cancer cells that harbor key driver mutations while leaving normal cells alone. Targeting cells that contain these mutations is often challenging because many common mutations exist in driver genes that code for intracellular proteins that are not directly accessible to immunotherapies. The feasibility of targeting cancer-specific mutations that are presented as mutant peptides bound to the major histocompatibility complex (pMHC) of cells has been demonstrated. Targeting cells that present these mutant peptides using bispecific antibodies or chimeric antigen receptor (CAR) T cells provides a genetically specific therapy to identify cells that contain mutant proteins that would otherwise go undetected by immune surveillance.

[0016] Nonetheless, there remains a need for improved antibodies with high affinity to these mutant peptides.

[0017] Thus, described herein is a molecule comprising a first antigen-binding domain comprising: (i) an scFv light chain CDR1 comprising or consisting of SEQ ID NO:8; (ii) an scFv light chain CDR2 comprising or consisting of SEQ ID NO:9 or SEQ ID NO:23; (iii) an scFv light chain CDR3 comprising or consisting of SEQ ID NO:10; (iv) an scFV heavy chain CDR1 comprising or consisting of SEQ ID NO:17; (v) an scFV heavy chain CDR2 comprising or consisting of SEQ ID NO:18 or SEQ ID NO:27; and (vi) an scFV heavy chain CDR3 comprising or consisting of SEQ ID NO:19.

[0018] In some embodiments, the first antigen-binding domain comprises (a) an scFv light chain CDR2 comprising or consisting of SEQ ID NO:23, or (b) an scFv heavy chain CDR2 comprising or consisting of SEQ ID NO:27, or (c) an scFv light chain CDR2 comprising or consisting of SEQ ID NO:23 and an scFv heavy chain CDR2 comprising or consisting of SEQ ID NO:27.

[0019] In some embodiments, the first antigen-binding domain comprises (i) an scFv light chain comprising a sequence at least 90% identical to SEQ ID NO:7 or SEQ ID NO:22, and (ii) an scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO:16 or SEQ ID NO:26. In some embodiments, the first antigen-binding domain comprises (a) an scFv light chain comprising a sequence at least 90% identical to SEQ ID NO:7, and an scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO:26, or (b) an scFv light chain comprising a sequence at least 90% identical to SEQ ID NO:22, and an scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO:16, or (c) an scFv light chain comprising a sequence at least 90% identical to SEQ ID NO:22, and an scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO:26.

[0020] In some embodiments, the first antigen-binding domain comprises (i) an scFv light chain comprising a sequence at least 98% identical to SEQ ID NO:7 or SEQ ID NO:22, and (ii) an scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO:16 or SEQ ID NO:26. In some embodiments, the first antigen-binding domain comprises (a) an scFv light chain comprising a sequence at least 98% identical to SEQ ID NO:7, and an scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO:26, or (b) an scFv light chain comprising a sequence at least 98% identical to SEQ ID NO:22, and an scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO:16, or (c) an scFv light chain comprising a sequence at least 98% identical to SEQ ID NO:22, and an scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO:26.

[0021] In some embodiments, the first antigen-binding domain comprises (i) an scFv light chain comprising or consisting of SEQ ID NO:7 or SEQ ID NO:22, and (ii) an scFv heavy chain comprising or consisting of SEQ ID NO:16 or SEQ ID NO:26. In some embodiments, the first antigen-binding domain comprises (a) an scFv light chain comprising or consisting of SEQ ID NO:7, and an scFv heavy chain comprising or consisting of SEQ ID NO:26, or (b) an scFv light chain comprising or consisting of SEQ ID NO:22, and an scFv heavy chain comprising or consisting of SEQ ID NO:16, or (c) an scFv light chain comprising or consisting of SEQ ID NO:22, and an scFv heavy chain comprising or consisting of SEQ ID NO:26.

[0022] In some embodiments, the molecule is selected from the group consisting of an antibody, an antibody fragment, a single chain variable fragment (scFv), a chimeric antigen receptor (CAR), a T cell receptor (TCR), a TCR mimetic, a tandem scFv, a bispecific T cell engager, a diabody, a single chain diabody (scDb), a scFv-Fc, a bispecific antibody, and a dual affinity retargeting antibody (DART).

[0023] In some embodiments, the molecule further comprises a second antigen binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27. In some embodiments, the second antigen binding domain is capable of binding to CD3. In some embodiments, the second antigen binding domain capable of binding to CD3 comprises a variable light chain and a variable heavy chain selected from those shown in Table 3. In some embodiments, the second antigen binding domain capable of binding to CD3 comprises or consists of any one of those shown in Table 2 (SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51).

[0024] In some embodiments, the molecule is a single chain diabody (scDb). In some embodiments, the single chain diabody comprises, in order from N-terminus to C-terminus, (i) an scFv light chain comprising (a) an scFv light chain CDR1 comprising or consisting of SEQ ID NO:8, (b) an scFv light chain CDR2 comprising or consisting of SEQ ID NO:9 or SEQ ID NO:23, (c) an scFv light chain CDR3 comprising or consisting of SEQ ID NO:10, (ii) an antigen binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27, and (iii) an scFv heavy chain comprising (a) an scFv heavy chain CDR1 comprising or consisting of SEQ ID NO:17, (b) an scFv heavy chain CDR2 comprising or consisting of SEQ ID NO:18 or SEQ ID NO:27, and (c) an scFv heavy chain CDR3 comprising or consisting of SEQ ID NO:19.

[0025] In some embodiments, the single chain diabody comprises (a) an scFv light chain CDR2 that comprises or consists of SEQ ID NO:23, or (b) an scFv heavy chain CDR2 that comprises or consists of SEQ ID NO:27, or (c) an scFv light chain CDR2 that comprises or consists of SEQ ID NO:23 and an scFv heavy chain CDR2 that comprises or consists of SEQ ID NO:27. In some embodiments, the single chain diabody comprises, in order from N-terminus to C-terminus, (i) an scFv light chain that comprises or consists of SEQ ID NO:7 or SEQ ID NO:22, (ii) an antigen binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27, and (iii) an scFv heavy chain that comprises or consists of SEQ ID NO:16 or SEQ ID NO:26. In some embodiments, the single chain diabody comprises (a) an scFv light chain comprising or consisting of SEQ ID NO:7, and an scFv heavy chain comprising or consisting of SEQ ID NO:26, or (b) an scFv light chain comprising or consisting of SEQ ID NO:22, and an scFv heavy chain comprising or consisting of SEQ ID NO:16, or (c) an scFv light chain comprising or consisting of SEQ ID NO:22, and an scFv heavy chain comprising or consisting of SEQ ID NO:26.

[0026] In some embodiments, the antigen binding domain is a CD3 antigen binding domain. In some embodiments, the CD3 antigen binding domain comprises a variable light chain and a variable heavy chain selected from those depicted in Table 3.

[0027] In some embodiments, the variable light and variable heavy chains of the antigen-binding domain are separated by a linker, preferably a 3xG4S linker (SEQ ID NO: 13). In some embodiments, the antigen-binding domain capable of binding to CD3 comprises or consists of any one of those shown in Table 2 (SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51).

[0028] In some embodiments, the molecule further comprises a first linker between the scFv light chain and the antigen binding domain, and a second linker between the antigen binding domain and the scFv heavy chain, In some embodiments, the first linker comprises or consists of G4S (SEQ ID NO:11) and the second linker comprises or consists of G4S (SEQ ID NO:15).

[0029] Also provided herein is a method for treating a mammal having a cancer expressing a mutant peptide comprising or consisting of HMTEVVRHC (SEQ ID NO: 1), the method comprising administering to the mammal a molecule according to any one of claims 1-24. In some embodiments, the mammal is a human. In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative disease, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, breast cancer, prostate cancer, esophageal cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, thyroid cancer, germ cell tumor, or melanoma.

[0030] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as each individual number within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. as well as each individual number within that range, e.g., 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0031] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0032] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or absent (e.g., detecting). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Evaluation can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent, depending on the context.

[0033] As used herein, the term "about" a number refers to a number plus or minus 10% of that number. The term "about" a range refers to a range of minus 10% of the lowest value and plus 10% of the highest value.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0035] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0036] [Figure 1] The design of the variant library is shown. The original H2 scFv was modified at 61 sites across all six CDRs with each of the 19 amino acids for a total of 1159 variants. The scFv library was directly synthesized and cloned into the pADL-10b vector for panning by phage display. [Diagram 2] The sites of amino acid modifications in picked panning colonies are indicated. SS320 competent cells were infected with phage pools from the end of the 4th and 5th rounds of panning and plated to pick single colonies. 100 colonies per round were subjected to Sanger sequencing to identify individual variants. High diversity remained at the end of panning, with few recurring variants and all CDRs represented in sequenced colonies. [Diagram 3]Results of screening variants with ammonium thiocyanate and urea are shown. Predicted structurally related phage clones were applied to ELISA plates coated with R175H / A2 pMHC or R175WT pMHC. After an initial wash, plates were treated with various concentrations of NH4SCN or urea to elute low affinity binders. Variants were assessed by linear regression analysis of ELISA absorbance (A450) versus wash concentration. Slopes are displayed for variants showing binding of R175H:R175WT over or above the original H2 variant. [Figure 4] The results of thiocyanate-based screening are shown for pooled phages. Phage pools from the fourth round of panning were screened with 0.5 M NH4SCN wash or control wash (HBSPE). Phages were applied to an ELISA plate coated with R175H / A2 monomer and washed with TBST followed by test wash buffer (SCN or HBSPE). Bound phages were eluted and used to infect SS320 cells to generate more phages. The same protocol was repeated with this single enriched phage pool. Variant enrichment was tracked using next-generation sequencing. Variants with a percentage of reads in the top 50th percentile and enriched at least 2-fold from the starting phage pool of the fourth round are shown. Selected enriched variants were then screened as scDbs as well as structurally predicted variants. [Diagram 5]Figure 1 shows increased relative binding of the variant scDbs to R175H / HLA-A2 monomers compared to the original H2 scDb. To confirm that the expressed scDbs specifically bind R175H / A2 monomers and can bind CD3, the scDbs were applied to plates coated with R175H / A2 monomers, R175WT / A2 monomers, or CD3δ / ε heterodimers. When normalized to the CD3 binding signal, most variant scDbs showed increased relative binding to R175H / HLA-A2 monomers by ELISA compared to the original H2-scDb. All variants had no R175WT / HLA-A2 binding. [Figure 6] Results are shown for testing the sensitivity of scDbs in peptide-pulsed co-cultures. TAP-deficient T2A3 cells were pulsed with various concentrations of the R175H 9mer or the R175WT 9mer. The scDbs were tested for their ability to activate T cells against peptide-pulsed cells at low pulse concentrations. T cells were co-cultured at a 2:1 effector:target cell ratio with 1 nM of scDb in all conditions. T cells produced high levels of IFNg against R175H-pulsed cells, but not with all scDbs in R175WT cells. The Y57I and F53S scDbs led to increased IFNg responses at 1 nM peptide compared to the double mutant and the parent H2 scDb. [Figure 7]Figure 1 shows that the H2 variants have a lower EC50 than the original H2 scDb in co-culture with KMS26 cells. 5x104 primary human T cells with endogenous p53R175H (TP53 R175H, filled symbols) or p53 knockout (TP53KO, open symbols) and 2.5x104 KMS26 target cells were co-cultured overnight in the presence of 3-fold dilutions of scDb from 10 nM to 0.169 pM. The H2 variants result in increased cytotoxicity (A) and interferon gamma responses (B) when compared to the original H2 scDb at the same concentrations of scDb. EC50 values ​​against the R175H cell line were determined by fitting a 5-parameter logistic curve (R175H solid line, p53KO dotted line). [Figure 8A] Figures 8A-8D show that the H2 variant produces strong cytotoxicity against target cell lines carrying the endogenous R175H mutation. The variant scDb was tested at 0.01 nM, 0.1 nM, and 1 nM in co-cultures of human T cells with endogenous R175H HLA-A2+ cell lines at an effector-to-target cell ratio of 2:1. In all target cell lines (TYKnu, A; KMS26, B; KLE, C; Nalm6, D), the variant scDb showed increased activity over the original H2 scDb. Toxicity at each scDb concentration was compared by ordinary two-way ANOVA with Tukey's multiple comparison test. Nalm6 R175H is an HLA-A2-positive cell line engineered to carry the TP53 R175H mutation by CRISPR editing. A-C: Bars for each scDb, from left to right: R175H, 0.01 nM; R175H, 0.1 nM; R175H, 1 nM; p53KO, 0.01 nM; p53KO, 0.1 nM; p53KO, 1 nM; Figure 8D: Bars for each scDb, from left to right: R175H, 0.01 nM; R175H, 0.1 nM; R175H, 1 nM; p53KO, 0.01 nM; p53KO, 0.1 nM; p53KO, 1 nM. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A] Figures 9A-9D show that F53S, Y57I and F53S / Y57I have increased affinity for R175H / HLA-A2 by SPR. The binding affinity of the original H2 (A), F53S (B), Y57I (C) and F53S / Y57I (D) scDbs was measured by surface plasmon resonance (SPR). Chips coated with R175H / A2 (dashed black line) or R175WT / A2 (dashed light grey line) monomers were exposed to increasing concentrations of the scDb. The measured affinities (KD) for the original H2 (A), F53S (B), Y57I (C) and F53S / Y57I (D) are 29.5 nM, 12.9 nM, 6.8 nM and 3.3 nM, respectively. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10A]Figures 10A-10I show that H2 variants can control tumor growth in vivo. A: To test whether the variant scDb improved efficacy in vivo compared to the original H2 scDb, 13-15 week old female NSG mice were inoculated with 1x106 luciferase positive KMS26 cells and 1x107 human T cells 2 days before treatment with a continuous infusion pump containing the scDb (scDb dose 0.15mg / kg / day for 14 days). Treatment groups were randomized on day -1. Tumor burden was tracked every 3 days by bioluminescence imaging using an IVIS system. N=6 mice per group. B-F: Total flux measurements were normalized to the injected control fluorochrome measured across the chest for each mouse. B, original H2; C, F53S; D, Y57I; E, F53S / Y57I; F: L2 isotype control. G: Bioluminescence images at each time point are shown. Luminescence was detected over a 60 second exposure time at all time points. H, Y57I and F53S / Y57I control KMS26 tumor growth in vivo. Two-way repeated measures ANOVA with Geissner-Greenhouse correction and Dunnett's multiple comparison test comparing each treated scDb to the original H2 scDb at each time point show that Y57I and F53S / Y57I control tumor growth better than H2 on days 5 and 8 of treatment (P<0.05). All flux measurements were normalized to the injected control fluorochrome measured across the chest for each mouse. I, Y57I control KMS26 tumor growth in vivo. Mann-Whitney multiple test of bioluminescence data from Figures 9A-9G shows that Y57I improved tumor control on day 8 compared to the original H2 scDb (P=0.019). Two weeks after treatment, no tumor recurrence was observed in mice treated with Y57I. N=6 mice per group. All flux measurements were normalized to the injected control fluorochrome measured across the thorax for each mouse. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 10F] See legend to Figure 10A. [Figure 10G] See legend to Figure 10A. [Figure 10H] See legend to Figure 10A. [Figure 10I] See legend to Figure 10A. [Figure 11A] Figures 11A-11G show that the H2 variant controls Nalm6R175H tumor growth in vivo. A: To test whether the H2 variant can better control the faster growing Nalm6R175H cell line at lower scDb doses, 7-8 week old female NSG mice were inoculated with 5x105 luciferase positive Nalm6R175H cells and 1x107 human T cells 2 days before treatment with a continuous infusion pump containing scDb (scDb dose 0.075mg / kg / day for 14 days). Treatment groups were randomized on day -1. Tumor burden was tracked by bioluminescence imaging using an IVIS system. Two independent experiments with N=6 per scDb were performed. Results are reported in aggregate, N=12 total. B-F: Mean brightness values: B, original H2; C, F53S; D, Y57I; E, F53S / Y57I; F: isotype control. G: Two-way repeated measures ANOVA with Geissner-Greenhouse correction and Dunnett's multiple comparison test show that at day 7 of treatment, all H2 variants improved tumor control (H2 vs. F53S P=0.0093, Y57I P=0.0126, F53S / Y57I P=0.0030), with H2 outperforming the isotype control scDb (P<0.0001). By Dunnett's multiple comparison test, F53S / Y57I showed superior tumor control compared to H2 at day 14 (two-way repeated measures ANOVA, P=0.0295). [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E]See legend to Figure 11A. [Figure 11F] See legend to Figure 11A. [Figure 11G] See legend to Figure 11A. [Figure 12A] Figures 12A-12E show that the Y57I scDb regulates KMS26 in an in vivo delayed treatment model. A: To compare the antitumor efficacy of scDb Y57I in an established tumor model, 7-9 week old female NSG mice were inoculated with 3.5x105 KMS26 cells and randomized 6 days later on treatment day -1. 7 days after tumor inoculation, mice were treated intravenously with 1x107 human T cells and treated with 0.075 mg / kg / day of the scDb using a continuous release pump surgically placed in the peritoneal cavity for 14 days, N=5 per group. B-D: Mean brightness values: B: original H2; C: Y57I, D: isotype. E: group mean brightness. At the end of treatment on day 14, Mann-Whitney tests comparing H2 scDb with Y57I scDb showed that mice treated with Y57I scDb had a lower tumor burden (P=0.0079). [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description This document relates to methods and materials for evaluating a mammal having or suspected of having cancer and / or for treating a mammal having cancer. For example, this document provides methods and materials for using molecules that include one or more antigen binding domains (e.g., single chain variable fragments (scFvs)) capable of binding to a modified peptide (e.g., a tumor antigen) to treat a mammal having cancer.

[0038] One example of a bispecific antibody targeting an intracellular driver gene is the TP53 R175H mutation, which is presented as a 9-mer peptide on HLA-A2 (mutant peptide TIFF2024546923000002.tif8165 Corresponding to positions 168 to 176 of SEQ ID NO: 3, with an R→H mutation at position 175);WT peptide TIFF2024546923000003.tif9165. See Hsiue et al., “Targeting a Neoantigen Derived from a Common TP53 Mutation,” Science 371(6533):eabc8697(2021). See also WO2021 / 12784, each of which is incorporated herein by reference in its entirety. The H2 bispecific antibody contains an anti-CD3 binding domain at one end of the molecule and a pMHC binding domain at the opposite end. Upon binding to target cell pMHC and CD3 in the T cell receptor complex of the T cell, the bispecific antibody activates the T cell to induce both cytokine release and T cell-mediated killing of the target cell. This H2 bispecific antibody can promote anti-cancer cell killing in vitro and limit tumor growth in vivo, but further improvements may allow for improved tumor clearance and performance in vivo. Indeed, others have shown that by increasing the binding affinity of bispecific antibodies, their potency can be significantly improved.

[0039] In particular, affinity matured H2 variants that maintain specificity for the R175H mutant peptide with improved performance both in vitro and in vivo, identified by a screening method to select variants containing a single amino acid change in the pMHC binding domain and then distinguish specific variants by relative affinity. The variants with high relative affinity identified by this method outperform the original H2 variant both in vitro and in vivo. These variant bispecific antibodies have higher affinity as measured by surface plasmon resonance (SPR) while maintaining the same specificity for the R175H mutant peptide. These affinity matured H2 variants can also be adapted for use as chimeric antigen receptors (CARs) or other scFv-based therapeutics.

[0040] Methods and compositions for evaluating a mammal having or suspected of having cancer and / or treating a mammal having cancer are also described herein. For example, one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of targeting (e.g., binding to) the TP53 R175H mutant peptide can be used to evaluate a mammal having or suspected of having cancer and / or treat a mammal having cancer (e.g., a cancer expressing one or more modified peptides). In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of targeting (e.g., binding to) the TP53 R175H mutant peptide can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal having or suspected of having cancer. In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of targeting (e.g., binding to) the TP53 R175H mutant peptide can be administered to a mammal having cancer (e.g., a cancer expressing a modified peptide) to treat the mammal.

[0041] As used herein, a modified peptide is a peptide derived from a modified polypeptide. A modified polypeptide can be any suitable modified polypeptide (e.g., a polypeptide having a disease-causing mutation, such as a mutation in an oncogenic gene or a mutation in a tumor suppressor gene). A modified peptide can have one or more amino acid modifications (e.g., substitutions) relative to a WT peptide (e.g., a peptide derived from the WT polypeptide from which the modified polypeptide is derived). A modified peptide can also be referred to as a mutant peptide. In some cases, a modified peptide can be a tumor antigen. Examples of tumor antigens include, but are not limited to, MANA, tumor-associated antigens, and tumor-specific antigens.

[0042] The modified peptide can be of any suitable length. In some cases, the modified peptide can be about 7 amino acids to about 25 amino acids in length (e.g., about 8 amino acids to about 25 amino acids, about 9 amino acids to about 25 amino acids, about 10 amino acids to about 25 amino acids, about 11 amino acids to about 25 amino acids, about 12 amino acids to about 25 amino acids, about 13 amino acids to about 25 amino acids, about 15 amino acids to about 25 amino acids, about 18 amino acids to about 25 amino acids, about 20 amino acids to about 25 amino acids, about 7 amino acids to about 22 amino acids, about 7 amino acids to about 20 amino acids, about 7 amino acids to about 18 amino acids, about 7 amino acids to about 15 amino acids, about 7 amino acids to about 12 amino acids, about 7 amino acids to about 10 amino acids, about 7 amino acids to about 9 amino acids, about 8 amino acids to about 22 amino acids, about 10 amino acids to about 18 amino acids, about 12 amino acids to about 15 amino acids, about 8 amino acids to about 12 amino acids, about 12 amino acids to about 18 amino acids, about 18 amino acids to about 22 amino acids, or about 9 amino acids to about 10 amino acids). For example, the modified peptide can be about 9 amino acids long. For example, the modified peptide can be about 10 amino acids long. The modified peptide can be derived from any modified polypeptide. The modified polypeptide from which the modified peptide described herein can be derived includes, but is not limited to, p53.

[0043] The modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be in complex with any suitable HLA. The HLA can be any suitable HLA allele. In some cases, the HLA can be a class I HLA (e.g., HLA-A, HLA-B, and HLA-C) allele. In some cases, the HLA can be a class II HLA (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR) allele. Examples of HLA alleles to which the modified peptides described herein can be complexed include, but are not limited to, HLA-A1 and HLA-A2. Exemplary HLA alleles for certain modified peptides are shown in Table 1. For example, the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be in complex with HLA-A2 and β2M.

[0044] Described herein are molecules that comprise one or more antigen binding domains (e.g., scFvs) capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). In some cases, a molecule that comprises one or more antigen binding domains capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) does not target (e.g., does not bind to) an uncomplexed modified peptide described herein (e.g., comprises or consists of a modified peptide described herein that is not present in a complex (e.g., a peptide-HLA-β2M complex). In some cases, a molecule that comprises one or more antigen binding domains capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprises or consists of SEQ ID NO:1) does not target (e.g., does not bind to) a WT peptide (e.g., a peptide derived from the WT polypeptide from which the modified polypeptide is derived).

[0045] Molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be any suitable type of molecule. In some cases, the molecules can be monovalent molecules (e.g., comprising a single antigen binding domain). In some cases, the molecules can be multivalent molecules (e.g., comprising two or more antigen binding domains and targeting two or more antigens simultaneously). For example, a bispecific molecule can comprise two antigen binding domains, a trispecific molecule can comprise three antigen binding domains, a tetraspecific molecule can comprise four antigen binding domains, etc. Examples of molecules containing an antigen-binding domain include, but are not limited to, antibodies, antibody fragments, scFvs, chimeric antigen receptors (CARs), T cell receptors (TCRs), TCR mimetics, tandem scFvs, bispecific T cell engagers, diabodies, scDbs, scFv-Fcs, bispecific antibodies, bispecific single-chain Fcs, dual affinity retargeting antibodies (DARTs), and any other molecule that includes at least one variable heavy chain (VH) and at least one variable light chain (VL). Any of these molecules can be used in accordance with the materials and methods described herein. In some cases, the antigen-binding domain can be an scFv. For example, a molecule that includes one or more antigen-binding domains (e.g., one or more scFvs) that can bind to the modified peptides described herein can be a CAR. For example, a molecule that includes two scFvs that can bind to the modified peptides described herein can be a single-chain diabody (scDb).

[0046] In some cases, when a molecule comprising one or more antigen-binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein is a multivalent molecule (e.g., a bispecific molecule), a first antigen-binding domain can bind to the modified peptide described herein, and a second antigen-binding domain can bind to an effector cell (e.g., an antigen present on an effector cell). Examples of effector cells include, but are not limited to, T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, plasma cells, macrophages, monocytes, microglia, dendritic cells, neutrophils, fibroblasts, and mast cells. Examples of antigens present on effector cells include, but are not limited to, CD3, CD4, CD8, CD28, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, CD27, Fc receptors (e.g., CD16a), and any other effector cell surface receptor. In some cases, the molecules described herein can include a first antigen-binding domain capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) and a second antigen-binding domain capable of binding to an antigen present on a T cell (e.g., CD3). In some cases, the sequence (e.g., scFv sequence) capable of binding to CD3 can be as shown in Table 2 or Table 3.In some cases, the sequence capable of binding to CD3 (e.g., an scFv sequence) can be as described elsewhere (see, e.g., Rodrigues et al., 1992 Int J Cancer Suppl. 7:45-50; Shalaby et al., 1992 J Exp Med. 175:217-25; Brischwein et al., 2006 Mol Immunol. 43:1129-43; Li et al., 2005 Immunology. 116:487-98; WO2012162067; US20070065437; US20070065437; US20070065437; US20070065437; and US20070065437). In some cases, the molecules described herein can include a first antigen binding domain capable of binding to a modified peptide described herein and a second antigen binding domain capable of binding to an antigen present on an NK cell (e.g., CD16a or NKG2D). In some cases, the sequence (e.g., scFv sequence) capable of binding to CD16a can be as shown in Table 4. By binding to both the modified peptide and the effector cell, the multivalent molecule can bring the cell expressing the modified peptide (e.g., as part of an HLA complex) into close proximity with the effector cell, allowing the effector cell to act on the cell expressing the modified peptide.

[0047] In some cases, when a molecule comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein is a multivalent molecule (e.g., a bispecific molecule), the molecule can be in any suitable format comprising at least one VH and at least one VL. For example, the VH and VL can be in any suitable orientation. In some cases, the VH can be N-terminal to the VL. In some cases, the VH can be C-terminal to the VL. In some cases, a linker amino acid sequence can be disposed between the VH and the VL.

[0048] In some cases, when the bispecific molecule comprises tandem scFvs, the tandem scFvs can be in any suitable orientation. Examples of tandem scFv orientations comprising scFv-A and scFv-B include, but are not limited to, VLA-LL-VHA-SL-VLB-LL-VHB, VLA-LL-VHA-SL-VHB-LL-VLB, VHA-LL-VLA-SL-VLB-LL-VHB, VHA-LL-VLA-SL-VHB-LL-VLB, VLB-LL-VHB-SL-VLA-LL-VHA, VLB-LL-VHB-SL-VHA-LL-VLA, VHB-LL-VLB-SL-VLA-LL-VHA, and VHB-LL-VLB-SL-VHA-LL-VLA, where SL is a short linker and LL is a long linker. The short linker can be from about 3 amino acids to about 10 amino acids in length. A short linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination.

[0049] In some cases, when the bispecific molecule is a diabody, the diabody can be in any suitable orientation. Examples of orientations of diabodies including scFv-A and scFv-B include, but are not limited to, VLA-SL-VHB and VLB-SL-VHA, VLA-SL-VLB and VHB-SL-VHA, VHA-SL-VLB and VHB-SL-VLA, VLB-SL-VHA and VLA-SL-VHB, VLB-SL-VLA and VHA-SL-VHB, and VHB-SL-VLA and VHA-SL-VLB, where SL is a short linker. The short linker can be about 3 amino acids to about 10 amino acids in length. The short linker can include any suitable amino acid (e.g., glycine and serine) in any suitable combination.

[0050] In some cases, when the bispecific molecule is a scDb, the scDb can be in any suitable orientation. Examples of orientations of scDbs, including scFv-A and scFv-B, include, but are not limited to, VLA-SL-VHB-LL-VLB-SL-VHA, VHA-SL-VLB-LL-VHB-SL-VLA, VLA-SL-VLB-LL-VHB-SL-VHA, VHA-SL-VHB-LL-VLB-SL-VLA, VLB-SL-VHA-LL-VLA-SL-VHB, VHB-SL-VLA-LL-VHA-SL-VLB, VLB-SL-VLA-LL-VHA-SL-VHB, and VHB-SL-VHA-LL-VLA-SL-VLB, where SL is a short linker and LL is a long linker. The short linker can be about 3 amino acids to about 10 amino acids in length. A short linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination.

[0051] In some cases, when the bispecific molecule is a scFv-Fc, the scFv-Fc can be in any suitable orientation. Examples of orientations of scFv-Fc-A, scFv-Fc-B, and scFv-Fc including Fc domains include, but are not limited to, VLA-LL-VHA-hinge-Fc and VLB-LL-VHB-hinge-Fc, VHA-LL-VLA-hinge-Fc and VHB-LL-VLB-hinge-Fc, VLA-LL-VHA-hinge-Fc and VHB-LL-VLB-hinge-Fc, VHA-LL-VLA-hinge-Fc and VLB-LL-VHB-hinge-Fc, where LL is a short linker. A long linker can be about 10 amino acids to about 25 amino acids in length. A long linker can include any suitable amino acid (e.g., glycine and serine) in any suitable combination. In some cases, the Fc domain of the scFv-Fc can include one or more modifications to increase heterodimerization and / or decrease homodimerization of the scFv-Fc. In some cases, the Fc domain in the scFv-Fc can exclude the hinge domain. In some cases, the Fc domain of the scFv-Fc can be at the N-terminus of the scFv.

[0052] In some cases, when the bispecific molecule is a bispecific single chain Fc, the bispecific single chain Fc can be in any suitable orientation. Examples of bispecific single chain Fc orientations include, but are not limited to, VLA-LL-VHA-SL-VHB-LL-VLB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VLA-LL-VHA-SL-VLB-LL-VHB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VHA-LL-VLA-SL-VLB-LL-VHB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VHA-LL-VLA-SL-VHB-LL-VLB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VHA-LL-VLA-SL-VHB-LL-VLB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, and VLA-SL-VHB-LL-VLB-VHA-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, where SL is a short linker and LL is a long linker. A short linker can be about 3 amino acids to about 8 amino acids in length. A short linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination. A long linker can be about 10 amino acids to about 25 amino acids in length. A long linker can include any suitable amino acids (e.g., glycine and serine) in any suitable combination. Any suitable Fc domain can be used in the bispecific single chain Fc. In some cases, the Fc domain can include an amino acid sequence derived from an IgG (e.g., a natural IgG). In some cases, the Fc domain can include an amino acid sequence that includes one or more modifications (e.g., one or more modifications to increase the stability of the molecule and / or to increase or decrease binding to one or more Fc receptors). In some cases, the Fc domain that can be used in the bispecific single chain Fc can exclude the hinge domain. In some cases, the Fc domain that can be used in the bispecific single chain Fc can be at the N-terminus of the scFv.In some cases, the Fc domains that can be used in the bispecific single chain Fc can be as described elsewhere (see, e.g., SEQ ID NOs:25-32 of International Patent Application Publication No. WO2017 / 134134A1; and, e.g., Table 38 and SEQ ID NOs:25-32 of International Patent Application Publication No. WO2017 / 134158A1).

[0053] Molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise any suitable complementarity determining region (CDR). For example, molecules comprising one or more antigen binding domains capable of binding to the modified peptides described herein can comprise a variable heavy chain (VH) having three VH complementarity determining regions (CDR-VH), and a variable light chain (VL) having three VL CDRs (CDR-VL).

[0054] In some cases, a molecule capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) comprises one or more mutations to H2 (SEQ ID NO:4) selected from the group consisting of Y57I, S92N, A31S, S95T, S92E, S95D, S95K, R24D, V29D, F53S, S26P, V29E, Q90D, and Y57L. In some cases, a molecule capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 14 mutations selected from the group consisting of Y57I, S92N, A31S, S95T, S92E, S95D, S95K, R24D, V29D, F53S, S26P, V29E, Q90D, and Y57L. In some cases, a molecule capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) comprises an F53S and / or Y57I mutation to H2 (SEQ ID NO:4). Thus, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the H2 CDRs (CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO:9, CDR-VL3: SEQ ID NO:10, CDR-VH1: SEQ ID NO:17, CDR-VH2: SEQ ID NO:18, and CDR-VH3: SEQ ID NO:19), provided that the molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 14 mutations (relative to H2, SEQ ID NO:4) selected from the group consisting of Y57I, S92N, A31S, S95T, S92E, S95D, S95K, R24D, V29D, F53S, S26P, V29E, Q90D, and Y57L. In some cases, the mutation(s) is F53S and / or Y57I.

[0055] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 9 or SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 18 or SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19, in this case, i) CDR-VL2 is SEQ ID NO: 23, or ii) CDR-VH2 is SEQ ID NO: 27, or iii) CDR-VL2 is SEQ ID NO: 23 and CDR-VH2 is SEQ ID NO: 27.

[0056] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 18 or SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19.

[0057] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 18, and CDR-VH3: SEQ ID NO: 19.

[0058] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19.

[0059] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 9 or SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19.

[0060] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 9, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19.

[0061] For example, a molecule capable of binding to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise one each of the CDRs set forth below: CDR-VL1: SEQ ID NO:8, CDR-VL2: SEQ ID NO: 23, CDR-VL3: SEQ ID NO: 10, CDR-VH1: SEQ ID NO: 17, CDR-VH2: SEQ ID NO: 27, and CDR-VH3: SEQ ID NO: 19.

[0062] In some cases, a molecule comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) may comprise any suitable set of CDR sequences (e.g., any of the sets of CDR sequences described herein).

[0063] Molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise any suitable sequence. For example, molecules capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can comprise, but are not limited to, the scFv sequence shown in any one of SEQ ID NO:20, SEQ ID NO:24, or SEQ ID NO:28. Examples of sequences (e.g., scFv sequences) capable of binding to specific modified peptides are shown in Table 1. In some cases, molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can have sequences that deviate from the sequences shown in Table 1, which may sometimes be referred to as variant sequences. For example, a molecule comprising one or more antigen binding domains capable of binding to the modified peptides described herein can have at least 75% sequence identity (e.g., at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or more) to any of the sequences shown in Table 1, so long as the variant sequence maintains the ability to bind to the modified peptides described herein. For example, a molecule comprising one or more antigen binding domains capable of binding to the modified peptides described herein can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) modifications (e.g., one or more amino acid substitutions) compared to the sequences shown in Table 1, so long as the variant sequence maintains the ability to bind to the modified peptides described herein. In some cases, a molecule comprising one or more antigen binding domains capable of binding to the modified peptides described herein can comprise any suitable set of CDR sequences described herein, and any sequence deviations from the sequences shown in Table 1 can be in the scaffold sequence(s).

[0064] A molecule comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be linked (e.g., covalently or non-covalently) to a label (e.g., a detectable label). The detectable label can be any suitable label. In some cases, the label can be used to aid in detecting the presence or absence of one or more modified peptides described herein. For example, a labeled molecule described herein can be used in vitro to detect cancer cells (e.g., cancer cells expressing a modified peptide described herein) in a sample obtained from a mammal. In some cases, the label (e.g., a detectable label) can be used to aid in determining the location of one or more modified peptides described herein. For example, a labeled molecule described herein can be used to monitor anti-tumor therapy and / or to detect cancer cells (e.g., cancer cells expressing a modified peptide described herein) in a mammal. Examples of labels that can be attached to the molecules described herein include, but are not limited to, radionuclides, contrast agents used in magnetic resonance imaging (MRI), computed tomography (CT), ultrasound (US), and other imaging modalities, chromophores, enzymes, and fluorescent molecules (e.g., green fluorescent protein and near infrared fluorescence).

[0065] Molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be conjugated (e.g., covalently or non-covalently) to a therapeutic agent. The therapeutic agent can be any therapeutic agent. In some cases, the therapeutic agent can be an anti-cancer agent. Examples of therapeutic agents that can be conjugated to the molecules described herein include, but are not limited to, anti-cancer agents such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, mertansine / emtansine (DM1), ravtansine / soraftansine (DM4), SN-38, calicheamicin, D6.5, dimeric pyrrolobenzodiazepines (PBD), alpha-amantin (AAMT), PNU-159682, ricin, Pseudomonas exotoxin A, diphtheria toxin, and gelonin.

[0066] The present specification also provides methods for using one or more molecules that include one or more antigen binding domains (e.g., scFv) that can bind to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). For example, one or more molecules that include one or more antigen binding domains (e.g., scFv) that can target (e.g., bind to) the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can be used to evaluate a mammal having or suspected of having cancer and / or treat a mammal having cancer (e.g., a cancer expressing the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). In some cases, one or more molecules that include one or more antigen binding domains that can bind to the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can be used to detect the presence or absence of the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) in a sample obtained from a mammal having or suspected of having cancer. In some cases, one or more molecules comprising one or more antigen binding domains capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered to a mammal having cancer (e.g., a cancer expressing the modified peptide) to treat the mammal. Administering one or more molecules comprising one or more antigen binding domains capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein to a mammal (e.g., a human) having cancer can be effective to treat the mammal.

[0067] Any type of mammal can be evaluated and / or treated as described herein. Examples of mammals that can be evaluated and / or treated as described herein include, but are not limited to, primates (e.g., humans and non-human primates, e.g., chimpanzees, baboons, or monkeys), dogs, cats, pigs, sheep, rabbits, mice, and rats. In some cases, the mammal can be a human.

[0068] The mammal can be evaluated and / or treated for any suitable cancer. In some cases, the cancer can express a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). The cancer can be a primary cancer. The cancer can be a metastatic cancer. The cancer can include one or more solid tumors. The cancer can include one or more non-solid tumors. Examples of cancers that may be assessed as described herein (e.g., based at least in part on the presence of one or more modified peptides described herein, e.g., comprising or consisting of) and / or treated as described herein (e.g., by administering one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified peptide described herein) include hematological cancers (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), multiple myeloma, MDS, and myeloproliferative disorders), lung cancer, pancreatic cancer, gastric cancer, colon cancer (e.g., colorectal cancer), ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, bone and soft tissue cancers (e.g., sarcoma), breast cancer, prostate cancer, esophageal cancer, stomach cancer, and the like. Cancers that may be present include, but are not limited to, renal cancer, head and neck cancer, brain cancer (e.g., glioblastoma multiforme and astrocytoma), thyroid cancer, germ cell tumors, and melanoma.

[0069] When assessing a mammal having or suspected of having cancer, one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be used to assess the presence or absence of one or more modified peptides described herein. For example, the presence, absence, or level of the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) in a sample obtained from a human can be used to determine whether the human has cancer. In some cases, the presence of the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) in a sample obtained from a mammal can be used to identify the mammal as having cancer. For example, if a sample obtained from a mammal has the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1), the mammal can be identified as having cancer.

[0070] Any suitable sample obtained from a mammal can be evaluated for the presence, absence, or level of the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). For example, biological samples such as tissue samples (e.g., breast tissue and cervical tissue from Papanicolaou (Pap) tests, etc.), fluid samples (e.g., blood, serum, plasma, urine, saliva, sputum, and cerebrospinal fluid), and solid samples (e.g., feces) can be obtained from a mammal and evaluated for the presence, absence, or level of the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). Any suitable method can be used to detect the presence, absence, or level of the modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1). For example, sequencing techniques including, but not limited to, Sanger sequencing, chemical sequencing, nanopore sequencing, sequencing by ligation (SOLiD sequencing), sequencing using mass spectrometry, whole exome sequencing, whole genome sequencing, and / or next generation sequencing can be used to determine the presence, absence, or levels of a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) in a sample obtained from a mammal.

[0071] When treating a mammal with cancer, one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered to the mammal with cancer to treat the mammal. In some cases, the mammal may have a cancer that expresses a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein. For example, one or more molecules comprising one or more antigen binding domains capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered to a mammal with cancer that expresses the modified peptide to treat the mammal. For example, one or more molecules comprising one or more scFvs (e.g., one or more scDbs) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered to a mammal with cancer that expresses the modified peptide to treat the mammal.

[0072] In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) can be administered to a mammal (e.g., a mammal having cancer) one or more times over a period ranging from several days to several weeks.

[0073] In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be formulated into a composition (e.g., a pharma- ceutically acceptable composition) for administration to a mammal (e.g., a mammal having cancer). For example, one or more antigen binding domains capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be formulated with one or more pharma- ceutically acceptable carriers (additives), excipients, and / or diluents. In some cases, the pharma- ceutically acceptable carriers, excipients, or diluents can be naturally occurring pharma- ceutically acceptable carriers, excipients, or diluents. In some cases, the pharma- ceutically acceptable carriers, excipients, or diluents can be non-naturally occurring (e.g., artificial or synthetic) pharma- ceutical acceptable carriers, excipients, or diluents.Examples of pharma- ceutically acceptable carriers, excipients, and diluents that can be used in the compositions described herein include sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses, such as microcrystalline cellulose and cellulose ethers, such as hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropylmethylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinylpyrrolidone (crospovinidone), carboxymethylcellulose, polyethylene-polyoxypropylene block polymers, and cross-linked sodium carboxymethylcellulose (croscarmellose sodium)), titanium dioxide, azo dyes, silica gel, fumes, and the like. Examples of suitable additives include, but are not limited to, silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, benzyl alcohol, lysine hydrochloride, trehalose dihydrate, sodium hydroxide, parabens (e.g., methylparaben and propylparaben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn starch. In some cases, the pharma- ceutically acceptable carrier, excipient, or diluent may be an anti-adhesive, binder, colorant, disintegrant, flavor (e.g., natural flavors such as fruit extracts or artificial flavors), glidant, lubricant, preservative, adsorbent, and / or sweetener.

[0074] Compositions (e.g., pharmaceutical compositions) comprising one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be formulated into any suitable dosage form. Exemplary dosage forms include solid or liquid forms, including, but not limited to, gums, capsules, tablets (e.g., chewable tablets and enteric coated tablets), suppositories, solutions, enemas, suspensions, liquids (e.g., sterile solutions), sustained release formulations, delayed release formulations, pills, powders, and granules.

[0075] Compositions comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) or intratumoral administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier for injection, for example, water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0076] A composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered to any suitable location using any suitable technique. A composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be administered locally (e.g., intratumorally) or systemically. For example, a composition provided herein can be administered locally by intratumoral administration (e.g., injection into a tumor) or by administration to a biological space infiltrated by a tumor (e.g., intraspinal, intracerebellar, intraperitoneal and / or pleural administration). For example, a composition provided herein can be administered systemically by oral or intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).

[0077] The effective dose may vary depending on the risk and / or severity of the cancer, the route of administration, the age and general health of the subject, the use of excipients, the possibility of co-administration with other therapeutic treatments such as the use of other drugs, and the judgment of the treating physician. An effective amount of a composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein may be any amount that treats the cancer present in the subject without causing significant toxicity to the subject. If a particular subject does not respond to a particular amount, the amount of one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein may be increased (e.g., 2-fold, 3-fold, 4-fold, or more). After this larger amount is administered, the mammal is monitored for both responsiveness to the treatment and toxic symptoms, and adjustments are made accordingly. The effective amount may remain constant or may be adjusted as a sliding scale or variable dose depending on the subject's response to the treatment. Various factors may affect the actual effective amount used for a particular application, for example, frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the actual effective amount administered.

[0078] The frequency of administration of one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be any frequency that effectively treats a mammal with cancer without causing significant toxicity to the mammal. For example, the frequency of administration of one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be about 2 to about 3 times per week to about 2 to about 3 times per year. In some cases, a subject with cancer can receive a single dose of one or more antibodies described herein. The frequency of administration of one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can remain constant or can be variable during the treatment period. A course of treatment with a composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein may include a drug holiday. For example, a composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein may be administered every other month for a period of two years, followed by a six-month drug holiday, and such a regimen may be repeated multiple times. As with the effective amount, various factors may affect the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the condition (e.g., cancer) may require increased or decreased frequency of administration.

[0079] The effective period for administering a composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to a modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein can be any period that effectively treats a cancer present in a mammal without causing significant toxicity to the mammal. In some cases, the effective period can vary from several months to several years. In general, the effective period for treating a mammal with cancer can be from about one or two months to five or more years. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the condition being treated.

[0080] In certain cases, cancer in a mammal can be monitored to evaluate the effectiveness of cancer treatment. Any suitable method can be used to determine whether to treat a mammal with cancer. For example, imaging techniques or laboratory assays can be used to evaluate the number of cancer cells and / or the size of tumors present in a mammal. For example, imaging techniques or laboratory assays can be used to evaluate the location of cancer cells and / or tumors present in a mammal.

[0081] In some cases, one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1) can be administered to a mammal having cancer as a combination therapy with one or more additional cancer therapies (e.g., anti-cancer agents). The cancer treatment can include any suitable cancer therapy. In some cases, the cancer treatment can include surgery. In some cases, the cancer treatment can include radiation therapy. In some cases, the cancer treatment can include administration of one or more therapeutic agents (e.g., one or more anti-cancer agents). Examples of anticancer drugs include platinum compounds (e.g., cisplatin or carboplatin), taxanes (e.g., paclitaxel, docetaxel, or albumin-bound paclitaxel, such as nab-paclitaxel), altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, and leuprolide), antiestrogens (e.g., tamoxifen), aromatase inhibitors (e.g., letrozole, anastrozole, and exemestane), angiogenesis inhibitors (e.g., bevacizumab), poly(ADP)-ribose polymerase (PARP) inhibitors (e.g., olaparib, rucaparib, and niraparib), radioactive phosphorus, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, IL-2 antibodies and other cytokines, other bispecific antibodies, and any combination thereof. When one or more molecules comprising one or more antigen binding domains (e.g., scFv) capable of binding to the modified TP53 R175H mutant peptide (e.g., comprising or consisting of SEQ ID NO:1) described herein are used in combination with one or more additional cancer therapeutics, the one or more additional cancer therapeutics can be administered simultaneously or independently.For example, a composition comprising one or more molecules comprising one or more antigen binding domains (e.g., scFvs) capable of binding to a modified TP53 R175H mutant peptide described herein (e.g., comprising or consisting of SEQ ID NO:1) may be administered first, and one or more additional cancer therapeutics may be administered second, or vice versa.

[0082] Also provided herein are kits that include one or more molecules that include one or more antigen binding domains (e.g., scFvs) that can bind to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1). For example, the kits can include compositions (e.g., pharma- ceutically acceptable compositions) that include one or more molecules that include one or more antigen binding domains (e.g., scFvs) that can bind to the modified TP53 R175H mutant peptides described herein (e.g., comprising or consisting of SEQ ID NO:1). In some cases, the kits can include instructions for carrying out any of the methods described herein. In some cases, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some cases, the kits can provide a means (e.g., a syringe) for administering any of the compositions (e.g., pharmaceutical compositions) described herein.

[0083] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0084] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0085] Example 1: Screening of single mutation variants for improved binding properties A phage display library consisting of 1159 single chain variable fragments (scFvs), each with a single amino acid change from the original H2 R175H-targeting scFv (SEQ ID NO: 4), was used to screen for improved variants targeting R175H / HLA-A2. A total of 61 sites across the six complementarity determining regions (CDRs) were included in the single mutant library with all 20 amino acids (excluding wild type) represented at each site (Figure 1). To identify clones that exhibited high specificity for R175H / HLA-A2 pMHC, five rounds of panning with negative selection against R175WT pMHC and a TP53 R175WT HLA-A2 positive cell line, followed by positive selection on R175H pMHC, were completed. Initial testing showed that after five rounds of panning, the phage pool contained variants present across all six CDRs and retained diversity (Figure 2).

[0086] Data from the crystal structure of H2 Fab bound to HLA-A2 were used to select panning clones with potential structural relevance for screening under more stringent binding conditions. Ammonium thiocyanate (NH4SCN) and urea washes were used to remove scFvs with weak binding to the R175H monomer and to identify clones specific for R175H over R175WT (Figure 3). Additional NH4SCN-based screening of pooled phages from the fourth and fifth rounds of panning identified several variants that were enriched in the thiocyanate wash (Figure 4).

[0087] Example 2: SCN-resistant variants have strong relative binding to R175H / HLA-A2 monomers The variants that maintained strong binding to the R175H monomer under stringent washing conditions were then converted to single-chain diabody (scDb) bispecific antibody format for functional testing. In turn, each scDb contains an IL-2 signal sequence (SEQ ID NO:6), a scFv light chain targeting pHLA (variant of SEQ ID NO:7), a GGGGS (G4S) linker (SEQ ID NO:11), an anti-CD3 scFv heavy chain (SEQ ID NO:12), a 3xG4S linker (SEQ ID NO:13), an anti-CD3 scFv light chain (SEQ ID NO:14), G4S (SEQ ID NO:15), a scFv heavy chain targeting pHLA (variant of SEQ ID NO:16), and a 5xHis affinity tag for purification. The scDbs were expressed in HEK293FT cells and purified using NiNTA resin.

[0088] To confirm that the expressed scDbs specifically bound R175H / A2 monomers and were capable of binding CD3, the scDbs were applied to plates coated with R175H / A2 monomers, R175WT / A2 monomers, or CD3δ / ε heterodimers. When normalized to the CD3 binding signal, most variant scDbs showed increased relative binding to R175H / HLA-A2 monomers compared to the parent H2-scDb by ELISA (Figure 5).

[0089] Example 3: Identification of variants with improved sensitivity and activity against R175H-HLA-A2 in vitro Three candidate variants, including a double mutant combining the two most potent single mutants (F53S (SEQ ID NO: 21), Y57I (SEQ ID NO: 25), and F53S / Y57I (SEQ ID NO: 29)), were functionally tested as scDbs in vitro and in vivo. Variant expression plasmids were generated by site-directed mutagenesis and used to express each scDb in HEK293FT cells. The scDbs for in vivo testing were produced on a larger scale and purified by size-exclusion chromatography. The variant scDbs were tested for their ability to activate human T cells in co-culture with TAP-deficient T2A3 cells pulsed with various levels of R175H or R175WT 9-mer peptide. Variants Y57I and F53S showed improved sensitivity at low peptide pulse concentrations (1 nM) compared to the original H2 scDb (Figure 6). All H2 variants showed increased cytotoxicity, interferon-γ responses, and lower EC than the parent H2 scDb in overnight cocultures with KMS26 cells and human T cells in the presence of various concentrations of the scDb. 50 The results showed that the KMS26-p53 R175H Cytotoxicity EC of F53S, Y57I, and F53S / Y57I against 50 The value is 3.061 × 10 for H2 scDb. -11 7.864×10 compared to M -12 M, 4.749×10 -12 M, and 4.154 x 10 -12 At low scDb concentrations (13.7 pM and 4.57 pM), regular two-way ANOVA with Tukey's multiple comparison test shows that all H2 variants had increased cytotoxicity compared to the H2 scDb (P<0.0001), with Y57I and F53S / Y57I resulting in increased interferon-γ compared to the H2 scDb (P<0.0001). The H2 variants also have improved activity in coculture against various HLA-A2 positive cell lines carrying the endogenous TP53 R175H mutation (Figures 8A-8D). Indeed, Y57I and F53S / Y57I show similar or higher activity against the KMS26 cell line at 10-fold lower bispecific antibody concentrations compared to the original H2 scDb.

[0090] Example 4: The variant scDb binds with higher affinity to the R175H / HLA-A2 complex. To determine whether the H2 variants bind to the R175H peptide-MHC complex with different affinities than the original H2 scDb, binding of the F53S, Y57I and F53S / Y57I scDbs to the R175H / A2 monomer was measured by surface plasmon resonance (SPR) (Figures 9A-D). All variants bound to the R175H pMHC complex with higher affinity than the original H2 scDb. The KDs of the variants are 12.9 nM, 6.8 nM and 3.3 nM for F53S, Y57I and F53S / Y57I compared to 29.5 nM for the original H2 scDb. The variants did not show increased binding to p53 WT / A2 pMHC.

[0091] Example 5: Variant scDb outperforms the original H2 scDb in vivo To compare the in vivo antitumor efficacy of the three H2 variants F53S, Y57I, and F53S / Y57I with the parent H2 scDb, 13-15 week-old female NSG mice (NOD scid IL2rg) were injected with 1 × 10 6 of KMS26 cells (TP53 R175H, HLA-A2) and 1 × 10 7of human T cells were inoculated intravenously. Two days later, mice were treated with 0.15 mg / kg / day of scDb using a continuous release pump surgically placed in the mouse peritoneal cavity for 14 days (Figure 10A-G). N=6 mice per group. Tumor burden was monitored by bioluminescence imaging. All flux measurements were normalized to the injected control fluorescent dye measured across the thorax for each mouse. Figure 10H shows that F53S, Y57I and F53S / Y57I improved tumor control compared to the original H2 scDb on day 2 of treatment, and Y57I and F53S / Y57I improved tumor control compared to the original H2 scDb on days 5 and 8 of treatment (P<0.05, two-way repeated measures ANOVA with Geissner-Greenhouse correction and Dunnett's multiple comparison test). Figure 10I shows that Y57I controlled KMS26 tumor growth in vivo. Mann-Whitney multiple range tests of bioluminescence data from Figures 10A-10G show that Y57I improved tumor control compared to the original H2 scDb at day 8 (P=0.019). Two weeks after treatment, no tumor recurrence was observed in mice treated with Y57I.

[0092] Because the variant scDb and the original H2 had antitumor efficacy against KMS26 at a dosing regimen of 0.15 mg / kg / day, the variant scDb was tested at a 50% lower dose for antitumor efficacy against a second tumor model, Nalm6, which has a faster growth rate. Luciferase-expressing Nalm6 cells were transfected with homozygous p53 R175H The mice were genetically modified using CRISPR to carry the mutation. 7-8 week-old NSG (NOD scid IL2rg) mice were injected with 5 × 10 5 Nalm6 R175H cells and 1 x 10 7of human T cells were inoculated intravenously (Figure 11A). Two days later, mice were treated with 0.075 mg / kg / day of scDb using a continuous release pump surgically placed in the mouse peritoneal cavity for 14 days (Figure 11B-G). Tumor burden was monitored by bioluminescence imaging. Bioluminescence data from two independent experiments (N=6 per scDb) were combined, for a total of N=12 per scDb. On day 7 of treatment, two-way repeated measures ANOVA with Geissner-Greenhouse correction for the H2 scDb and Dunnett's multiple comparison test showed that all H2 variants improved tumor control (H2 vs. F53S P=0.0093, Y57I P=0.0126, F53S / Y57I P=0.0030) and that H2 was superior to the isotype control scDb (P<0.0001). According to Dunnett's multiple comparison test, F53S / Y57I showed superior tumor control compared with H2 on day 14 (two-way repeated measures ANOVA, P = 0.0295).

[0093] To determine whether Y57I performs better than the original H2 scDb in established tumor models, the Y57I scDb was tested in a delayed treatment model using KMS26 cells. 7-9 week old NSG (NOD scid IL2rg) mice were inoculated with 3.5 × 10 5 The mice were inoculated intravenously with 1 × 10 luciferase-expressing KMS26 cells. Seven days later, the mice were 7 Mice were treated intravenously with 100 mg / kg / day of human T cells and treated with 0.075 mg / kg / day of the scDb using a continuous release pump surgically placed in the peritoneal cavity for 14 days (Figures 12A-E). Tumor burden was monitored by bioluminescence imaging. N=5 per group. At the end of treatment on day 14, Mann-Whitney tests showed that Y57I-treated mice had lower tumor burdens than mice treated with the original H2 scDb (P=0.0079).

[0094] array SEQ ID NO:1 P53 R175H mutant peptide HMTEVVRHC SEQ ID NO:2 P53 R175 wild type peptide HMTEVVRRC SEQ ID NO:3 p53 >sp|P04637|p53_Human cell tumor antigen p53 OS=Homo sapiens OX=9606 GN=TP53 PE=1 SV=4 MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIR VEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKKSKKGQSTSRHKKLMFKTEGPDSD SEQ ID NO: 4 H2 scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIKRTGGGSGGGGSGGG ASEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS SEQ ID NO:5 H2 scDb MYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVE IKGGGGSEVQLQQSGPELVKPGASMKISKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVT VSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKE VQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSSHHHHHH SEQ ID NO:6 H2 IL-2 signal sequence MYRMQLLSCIALSLALVTNS SEQ ID NO: 7 scFv light chain targeting H2 R175H peptide DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIK SEQ ID NO: 8 scFv light chain CDR1 targeting H2 R175H peptide RASQDVNTAVA SEQ ID NO: 9 scFv light chain CDR2 targeting H2 R175H peptide SAYFLYS scFv light chain CDR3 targeting SEQ ID NO: 10 H2 R175H peptide QQYSRYSPV SEQ ID NO:11 Linker GGGGS SEQ ID NO:12 H2 CD3-targeting heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTY NQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV SS SEQ ID NO:13 Linker GGGGSGGGGSGGGGS SEQ ID NO:14 H2 CD3-targeting light chain DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPS KFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK SEQ ID NO:15 Linker GGGGS SEQ ID NO: 16 scFv heavy chain targeting H2 R175H peptide EVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDYTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVS S SEQ ID NO: 17 scFv heavy chain CDRH1 targeting H2 R175H peptide FNVYASGMH SEQ ID NO: 18 scFv heavy chain CDRH2 targeting H2 R175H peptide VAKIYPDSDYTYY SEQ ID NO: 19 scFv heavy chain CDRH3 targeting H2 R175H peptide SRDSSFYYVYAM SEQ ID NO: 20 F53S scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIKRTGGGSGGGGSGGG ASEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS SEQ ID NO: 21 scDb targeting F53S R175H peptide (without signal sequence or His tag) MYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKV EIKGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTV TVSSGGGGSGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS SEQ ID NO: 22 scFv light chain targeting F53S R175H peptide DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIK SEQ ID NO: 23 scFv light chain CDR2 targeting F53S R175H peptide SAYSLYS SEQ ID NO: 24 Y57I scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIKRTGGGSGGGGSGGG ASEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDITYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS SEQ ID NO: 25 scDb targeting Y57I R175H peptide (without signal sequence or His tag) MYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKV EIKGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTV TVSSGGGGSGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDITYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS SEQ ID NO: 26 scFv heavy chain targeting Y57I R175H peptide EVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDITYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS scFv heavy chain CDR2 targeting SEQ ID NO: 27 Y57I R175H peptide VAKIYPDSDITYY SEQ ID NO: 28 F53S / Y57I scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKVEIKRTGGGSGGGGSGGG ASEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDITYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVS SEQ ID NO: 29 scDb targeting F53S / Y57I R175H peptide (without signal sequence or His tag) MYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAYSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSRYSPVTFGQGTKV EIKGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTV TVSSGGGGSGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNVYASGMHWVRQAPGKGLEWVAKIYPDSDITYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRDSSFYYVYAMDYWGQGTLVTVSS

[0095] Table 1. scFv targeting R175H peptide TIFF2024546923000004.tif114138

[0096] Table 2: Anti-human CD3 scFv sequences TIFF2024546923000005.tif66138TIFF2024546923000006.tif209138

[0097] Table 3: Anti-human CD3 scFv sequences. The affinities of selected anti-CD3 clones were collected from the literature. TIFF2024546923000007.tif195138TIFF2024546923000008.tif131138

[0098] Table 4: Anti-human CD16a scFv sequences TIFF2024546923000009.tif111138

[0099] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A molecule comprising a first antigen-binding domain comprising variable light chain complementarity determining regions CDR-VL1, CDR-VL2, and CDR-VL3, and variable heavy chain complementarity determining regions CDR-VH1, CDR-VH2, and CDR-VH3, (i) CDR-VL1 comprises SEQ ID NO:8, CDR-VL2 comprises SEQ ID NO:9, CDR-VL3 comprises SEQ ID NO:10, CDR-VH1 comprises SEQ ID NO:17, CDR-VH2 comprises SEQ ID NO:27, and CDR-VH3 comprises SEQ ID NO:19; or (ii) CDR-VL1 comprises SEQ ID NO:8, CDR-VL2 comprises SEQ ID NO:23, CDR-VL3 comprises SEQ ID NO:10, CDR-VH1 comprises SEQ ID NO:17, CDR-VH2 comprises SEQ ID NO:18, and CDR-VH3 comprises SEQ ID NO:19; or (iii) CDR-VL1 comprises SEQ ID NO:8, CDR-VL2 comprises SEQ ID NO:23, CDR-VL3 comprises SEQ ID NO:10, CDR-VH1 comprises SEQ ID NO:17, CDR-VH2 comprises SEQ ID NO:27, and CDR-VH3 comprises SEQ ID NO:19; and wherein the first antigen-binding domain is capable of binding to a p53 R175H-HLA2 monomer, and the p53 R175H peptide comprises or consists of the peptide of SEQ ID NO:

1. molecule.

2. the first antigen-binding domain comprises: (i) an scFv light chain comprising a sequence at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 22; and (ii) an scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 16 or SEQ ID NO: 26 The molecule of claim 1 , comprising:

3. the first antigen-binding domain comprises: (a) the scFv light chain comprising a sequence at least 90% identical to SEQ ID NO: 7, and the scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 26; or (b) the scFv light chain comprising a sequence at least 90% identical to SEQ ID NO: 22, and the scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 16; or (c) the scFv light chain comprising a sequence at least 90% identical to SEQ ID NO: 22, and the scFv heavy chain comprising a sequence at least 90% identical to SEQ ID NO:

26. The molecule of claim 2 comprising:

4. the first antigen-binding domain comprises: (i) an scFv light chain comprising a sequence at least 98% identical to SEQ ID NO: 7 or SEQ ID NO: 22; and (ii) an scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO: 16 or SEQ ID NO: 26 The molecule of any one of claims 1 to 3, comprising:

5. the first antigen-binding domain comprises: (a) the scFv light chain comprising a sequence at least 98% identical to SEQ ID NO: 7, and the scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO: 26; or (b) the scFv light chain comprising a sequence at least 98% identical to SEQ ID NO: 22, and the scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO: 16; or (c) the scFv light chain comprising a sequence at least 98% identical to SEQ ID NO: 22, and the scFv heavy chain comprising a sequence at least 98% identical to SEQ ID NO:

26.

5. The molecule of claim 4, comprising:

6. the first antigen-binding domain comprises: (i) an scFv light chain comprising or consisting of SEQ ID NO: 7 or SEQ ID NO: 22, and (ii) an scFv heavy chain comprising or consisting of SEQ ID NO: 16 or SEQ ID NO: 26 The molecule of any one of claims 1 to 5, comprising:

7. the first antigen-binding domain comprises: (a) the scFv light chain comprising or consisting of SEQ ID NO: 7, and the scFv heavy chain comprising or consisting of SEQ ID NO: 26; or (b) the scFv light chain comprising or consisting of SEQ ID NO: 22, and the scFv heavy chain comprising or consisting of SEQ ID NO: 16; or (c) the scFv light chain comprising or consisting of SEQ ID NO: 22, and the scFv heavy chain comprising or consisting of SEQ ID NO:

26. The molecule of claim 6, comprising:

8. 8. The molecule of any one of claims 1 to 7, wherein the molecule is selected from the group consisting of an antibody, a single chain variable fragment (scFv), a chimeric antigen receptor (CAR), a T cell receptor (TCR), a TCR mimetic, a tandem scFv, a bispecific T cell engager, a diabody, a single chain diabody (scDb), a scFv-Fc, a bispecific antibody, and a dual affinity retargeting antibody (DART).

9. 9. The molecule of any one of claims 1 to 8, further comprising a second antigen-binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27.

10. The molecule of claim 9, wherein the second antigen-binding domain is capable of binding to CD3.

11. 11. The molecule of claim 10, wherein the second antigen-binding domain capable of binding to CD3 comprises a variable light chain and a variable heavy chain selected from those shown in Table 3.

12. 12. The molecule of claim 11, wherein the second antigen-binding domain capable of binding to CD3 comprises or consists of any one of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:

51.

13. 13. The molecule of any one of claims 1 to 12, which is a single chain diabody (scDb).

14. The single chain diabody comprises, in order from N-terminus to C-terminus: (i) (a) an scFv light chain CDR1 comprising or consisting of SEQ ID NO:8; (b) an scFv light chain CDR2 comprising or consisting of SEQ ID NO: 9 or SEQ ID NO: 23; (c) an scFv light chain CDR3 comprising or consisting of SEQ ID NO: 10 an scFv light chain comprising: (ii) an antigen-binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27; (iii) (a) an scFv heavy chain CDR1 comprising or consisting of SEQ ID NO: 17; (b) an scFv heavy chain CDR2 comprising or consisting of SEQ ID NO: 18 or SEQ ID NO: 27; and (c) an scFv heavy chain CDR3 comprising or consisting of SEQ ID NO: 19 an scFv heavy chain comprising: Including, the single chain diabody (a) the scFv light chain CDR2 comprising or consisting of SEQ ID NO: 23; or (b) the scFv heavy chain CDR2 comprising or consisting of SEQ ID NO: 27; or (c) the scFv light chain CDR2 comprising or consisting of SEQ ID NO: 23, and the scFv heavy chain CDR2 comprising or consisting of SEQ ID NO:

27.

14. The molecule of claim 13, comprising:

15. The single chain diabody comprises, in order from N-terminus to C-terminus: (i) an scFv light chain comprising or consisting of SEQ ID NO: 7 or SEQ ID NO: 22; (ii) an antigen-binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27; (iii) an scFv heavy chain comprising or consisting of SEQ ID NO: 16 or SEQ ID NO: 26; 15. The molecule of claim 13 or 14, comprising:

16. the single chain diabody (a) the scFv light chain comprising or consisting of SEQ ID NO: 7, and the scFv heavy chain comprising or consisting of SEQ ID NO: 26; or (b) the scFv light chain comprising or consisting of SEQ ID NO: 22, and the scFv heavy chain comprising or consisting of SEQ ID NO: 16; or (c) the scFv light chain comprising or consisting of SEQ ID NO: 22, and the scFv heavy chain comprising or consisting of SEQ ID NO:

26.

16. The molecule of claim 15, comprising:

17. The molecule of claim 14 or 15, wherein the antigen-binding domain is a CD3 antigen-binding domain.

18. 18. The molecule of claim 17, wherein the CD3 antigen binding domain comprises a variable light chain and a variable heavy chain selected from those shown in Table 3.

19. 19. The molecule of claim 18, wherein the variable light chain and the variable heavy chain of the antigen-binding domain are separated by a linker.

20. 20. The molecule of any one of claims 17 to 19, wherein the antigen binding domain capable of binding to CD3 comprises or consists of any one of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:

51.

21. a first linker between the scFv light chain and the antigen-binding domain; and A second linker between the antigen binding domain and the scFv heavy chain. The molecule of any one of claims 14 to 20, further comprising:

22. HMTEVVRHC (SEQ ID NO: 1) 1. A method for treating a mammal having cancer that expresses a mutant peptide comprising or consisting of: The method comprising administering to the mammal a molecule according to any one of claims 1 to 21.

23. 23. The method of claim 22, wherein the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative disorder, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, breast cancer, prostate cancer, esophageal cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, thyroid cancer, germ cell tumor, or melanoma.

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