Compositions and methods for treating neoplasia

Abstract

Provided and featured are T cell enhancers useful in improving patient immune responses against tumors, cancers and neoplasias.

Description

[0001] Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0002] COMPOSITIONS AND METHODS FOR TREATING NEOPLASIA

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to and the benefit of U.S. App. No. 63 / 716,394, filed November 5, 2024, and U.S. App. No. 63 / 728,062, filed December 4, 2024, each of which is hereby incorporated by reference in its entirety.

[0005] BACKGROUND

[0006] Immune cells play an important role in eliminating abnormal cells; however, immunity against tumor and tumor cells is often absent in patients with cancer. In such patients, therapies supporting anti-tumor immunity fail to work. Improved methods of increasing immunity against tumor cells in patients having cancer are urgently required.

[0007] SUMMARY

[0008] Described and provided herein are T cell enhancers useful in improving patient immune responses against cancers, including tumors and neoplasias.

[0009] In an aspect, the present disclosure provides a T cell engager (TCE) including a T cell binding domain or an antigen binding fragment thereof and aT cell receptor (TCR) component that specifically binds to a tumor suppressor protein 53 (TP53) polypeptide including a mutation presented by a major histocompatibility complex (MHC) molecule binding domain.

[0010] In another aspect, the present disclosure provides a method of eliciting an immune response against a tumor cell in a subject. The method involves administering to the subject the TCE of any of the above aspects, or embodiments thereof.

[0011] In another aspect, the present disclosure provides a method of activating a T cell. The method involves contacting the T cell with the TCE of any of the above aspects, or embodiments thereof.

[0012] In another aspect, the present disclosure provides T cell engager (TCE) including a CD3 binding domain or an antigen binding fragment thereof, and a T cell receptor (TCR) component, where the T cell receptor component specifically binds a tumor suppressor protein 53 (TP53) polypeptide or fragment thereof including an R175H mutation presented by a major histocompatibility complex (MHC) molecule.

[0013] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0014] In another aspect, the present disclosure provides a polynucleotide encoding the TCE of any of the above aspects, or embodiments thereof.

[0015] In another aspect, the present disclosure provides a vector including the polynucleotide of any of the above aspects, or embodiments thereof.

[0016] In another aspect, the present disclosure provides a cell including the polynucleotide or the vector of any of the above aspects, or embodiments thereof.

[0017] In another aspect, the present disclosure provides method of eliciting an immune response against a tumor cell in a subject. The method involves administering to the subject a TCE, or a vector of any of the above aspects, or embodiments thereof.

[0018] In another aspect, the present disclosure provides a method of activating a T cell. The method involves contacting the T cell with the TCE of any of the above aspects, or embodiments thereof.

[0019] In another aspect, the present disclosure provides a pharmaceutical composition including a TCE or vector of any of the above aspects, or embodiments thereof, and a pharmaceutical excipient.

[0020] In another aspect, the present disclosure provides a kit for immunotherapy. The kit includes the TCE or the vector of any of the above aspects, or embodiments thereof.

[0021] In another aspect, the present disclosure provides a method of treating a neoplasia in a subject. The method involves administering to the subject the TCE or the vector of any of the above aspects, or embodiments thereof.

[0022] In any of the above aspects, or embodiments thereof, the TP53 mutation is TP53 R175H.

[0023] In any of the above aspects, or embodiments thereof, the MHC molecule includes human leukocyte antigen (HLA) serotype A2. In any of the above aspects, or embodiments thereof, the HLA serotype is HLA-A*02:01.

[0024] In any of the above aspects, or embodiments thereof, the TCR component is derived from an affinity -matured TCR.

[0025] In any of the above aspects, or embodiments thereof, the T cell binding domain includes a CD3 binding domain. In any of the above aspects, or embodiments thereof, the CD3 binding domain includes an antigen binding domain of an anti-CD3 antibody.

[0026] In any of the above aspects, or embodiments thereof, the TCE further includes a halflife extension moiety. In any of the above aspects, or embodiments thereof, the half-life extension moiety is an Fc domain.

[0027] 2

[0028] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0029] In any of the above aspects, or embodiments thereof, the tumor cell is a solid tumor cell.

[0030] In any of the above aspects, or embodiments thereof, the tumor cell expresses a tumor associated antigen.

[0031] In any of the above aspects, or embodiments thereof, the tumor cell expresses an oncogenic driver mutation. In any of the above aspects, or embodiments thereof, the oncogenic driver mutation is a mutation in TP53.

[0032] In any of the above aspects, or embodiments thereof, the tumor cell expresses HLA serotype A2. In any of the above aspects, or embodiments thereof, the tumor cell expresses HLA serotype HLA-A*02:01.

[0033] In any of the above aspects, or embodiments thereof, the TCE includes a single polypeptide chain including the TCR component linked to the CD3 binding domain or antigen binding fragment thereof.

[0034] In any of the above aspects, or embodiments thereof, the CD3 binding domain is an anti-CD3 antibody or antigen-binding fragment thereof.

[0035] In any of the above aspects, or embodiments thereof, the CD3 binding domain includes or consists of a heavy’ chain variable (Vn) region and a light chain variable (VL) region of an anti-CD3 antibody.

[0036] In any of the above aspects, or embodiments thereof, the CD3 binding domain does not include a constant region of an anti-CD3 antibody.

[0037] In any of the above aspects, or embodiments thereof, the CD3 binding domain includes a Vn region of an anti-CD3 antibody comprising an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SS.

[0038] In any of the above aspects, or embodiments thereof, the CD3 binding domain includes a VL region of an anti-CD3 antibody comprising an amino acid sequence having at least 85% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFS GSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK.

[0039] In any of the above aspects, or embodiments thereof, the CD3 binding domain includes an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY

[0040] 3

[0041] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0042] NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK.

[0043] In any of the above aspects, or embodiments thereof, the TCE includes an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGG GGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVYWYRQSPDQGLQFLIQYYNGEERGIGNI LERFSAQQFPDLHSELNISSLELGDSALYFCASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGG GSGGGGSGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYS NGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIPNG GGGSDYKDDDDKHHHHHH.

[0044] In any of the above aspects, or embodiments thereof, the TCE includes an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGG GGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVYWYRQSPDQGLQFLIQYYNGEERGIGNI LERFSAQQFPDLHSELNISSLELGDSALYFCASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGG GSGGGGSGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYS NGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIPNG GGGSDYKDDDDKHHHHHH.

[0045] In any of the above aspects, or embodiments thereof, the TCE includes an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGG GGSDSGVTQTPKHLIAATGQRVTLRCTPRSGDLSVYWYQQSPDQGLQFLIQYYNGEERGIGN IPERFSAQQFPDLHSELNLSSLELGDSALYFCASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGG GGSGGGGSGGGSQKEVEQNSGPLSVPEGANASLNCTYSDRGSQSFFWYRQYSGKSPELIMSI YSNGDKDDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIP NGGGGSDYKDDDDKHHHHHH.

[0046] 4

[0047] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0048] In any of the above aspects, or embodiments thereof, the TCE includes an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGG GGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVYWYKQSPDQGLQFLIQYYNGEERGIGNI PERFSAQQFPDLHSELNLSSLELGDSASYFCASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGG GGSGGGGSGGGSQKEVEQNSGPLSVPEGAISSLNCTYSDRGSQSFFWYRQYSGKSPELIMSIY SNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSNSNSRAYQLTFGKGTKLSVIPY GGGGSDYKDDDDKHHHHHH.

[0049] In any of the above aspects, or embodiments thereof, the TCE includes from N- terminus to C-terminus or from C-terminus to N-terminus, the CD3 binding domain or an antigen binding fragment thereof, and the T cell receptor (TCR) component.

[0050] In any of the above aspects, or embodiments thereof, the vector is a plasmid vector. In any of the above aspects, or embodiments thereof, the vector is a viral vector. In any of the above aspects, or embodiments thereof, the vector is an adeno-associated viral (AAV) vector.

[0051] In any of the above aspects, or embodiments thereof, the neoplasia is adenocarcinoma and / or myeloma

[0052] Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the aspects and embodiments described herein will be apparent from the detailed description and from the claims.

[0053] Definitions

[0054] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology7(Walker ed., 1988); The Glossary of Genetics, 5th Ed.. R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0055] By ‘‘agent” is meant a peptide, nucleic acid molecule, or small compound. In an embodiment, the agent is a tumor cell engager (“TCE”) as disclosed herein.

[0056] 5

[0057] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0058] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In an embodiment, the amelioration involves decreasing, suppressing, attenuating, diminishing, arresting, or stabilizing the development or progression of a neoplasia.

[0059] By "alteration" is meant a change (increase or decrease) in the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels.

[0060] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0061] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0062] By “complementary” is meant capable of pairing to form a double-stranded nucleic acid molecule or portion thereof. In one embodiment, an antisense molecule is in large part complementary to a target sequence. The complementarity need not be perfect, but may include mismatches at 1, 2, 3, or more nucleotides.

[0063] By “decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages.

[0064] “Detect” refers to identifying or determining the presence, absence, or amount of an analyte to be detected.

[0065] By "detectable label" is meant a composition, reagent, or agent that, when linked to a molecule of interest, renders the latter detectable, via spectroscopic, photochemical,

[0066] 6

[0067] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0068] By “disease’' is meant any condition, disorder, or pathology that damages or interferes with the normal function of a cell, tissue, or organ. In an embodiment, the disease is a neoplasia. In an embodiment, the disease is a neoplasia comprising cells expressing a tumor suppressor protein (TP53) mutation, such as TP53 R175H. In an embodiment, the disease is a neoplasia comprising cells expressing a human leukocyte antigen (HLA) serotype A2 (i.e., HLA-A*02). In some embodiments, the HLA serotype is HLA-A*02:01.

[0069] The term “expression” or “expressed” as used herein in reference to a gene means the process of generating a transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0070] By "effective amount" is meant the amount of a required to ameliorate, decrease, abate, reduce, diminish, or eliminate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present aspects and embodiments as described for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0071] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20,

[0072] 7

[0073] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 30, 40, 50, 60, 70, 80, 90, or 100, 200. 300, 400, 500, 600, 700. 800, 900. or 1000 nucleotides or amino acids.

[0074] A “host cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokary otic or eukary otic cells that have been genetically engineered to contain a cloned gene(s) in the chromosome or genome of the host cell.

[0075] "Hybridization" means hydrogen bonding, which may7be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary7nucleobases. For example, adenine and thymine are complementary' nucleobases that pair through the formation of hydrogen bonds.

[0076] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the aspects and embodiments is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity7and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0077] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the described aspects and embodiments is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA

[0078] 8

[0079] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0080] By an "isolated polypeptide" is meant a polypeptide of the described aspects and embodiments that has been separated from components that naturally accompany it. Typically , the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by weight, of a polypeptide as described herein. An isolated polypeptide as described herein may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0081] By “marker” is meant any protein, polynucleotide, or other analyte having an alteration in expression level or activity that is associated with a disease or disorder.

[0082] By "operably linked” refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. The described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means positioning the coding sequence such that the expression of the coding sequence is controlled by the promoter.

[0083] By “poly adenylation signal sequence” (poly (A) signal sequence) or “poly (A) tail” is meant a sequence of multiple adenosine monophosphates at the 3’-end of mRNA or cDNA. The poly(A) tail is particularly important for nuclear export, translation, and for stabilizing or protecting mRNA from nucleases.

[0084] By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A polynucleotide fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0085] By “positioned for expression” is meant that a polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and

[0086] 9

[0087] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 translation of the sequence (i. e. , facilitates the production of, for example, a recombinant protein described herein).

[0088] The term “promoter” as used herein refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be “inducible,” in which transcription is initiated in response to an inducing agent or, in contrast, a promoter may be “constitutive,” in which an inducing agent does not regulate the rate of transcription. A promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types.

[0089] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0090] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0091] By “reference” is meant a standard or control condition. In one embodiment, the reference is an untreated patient. In one embodiment, the reference is a T cell not contacted with a T cell engager of the present disclosure. In one embodiment, the reference is a neoplasia in an untreated patient.

[0092] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides or at least about 300 nucleotides or any integer thereabout or therebetween.

[0093] Nucleic acid molecules useful in the methods as described herein include any nucleic acid molecule that encodes a polypeptide as described herein, or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the

[0094] 10

[0095] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 methods of the described aspects and embodiments include any nucleic acid molecule that encodes a polypeptide as described herein, or a fragment thereof, e.g., a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will ty pically exhibit substantial identity7. Polynucleotides having “substantial identity'’ to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0096] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, and less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, and of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA. are well known to those skilled in the art.

[0097] Various levels of stringency are accomplished by combining these various conditions as needed. In an embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In an embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In an embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0098] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, w ash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the w ash steps w ill be less than about 30 mM NaCl and 3 mM trisodium citrate, and less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the w ash steps w ill

[0099] 11

[0100] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 ordinarily include a temperature of at least about 25° C. of at least about 42° C, and of at least about 68° C. In an embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In an embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In an embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology', Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0101] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity’ to a reference amino acid sequence (for example, any one of the amino acid sequences descnbed herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In an embodiment, such a sequence is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0102] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University7of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions ty pically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0103] By "specifically binds" is meant a capture molecule that recognizes and binds a polypeptide as described herein, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a

[0104] 12

[0105] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 polypeptide as described herein. Capture molecules include, for example, T cell receptors, antibodies, or antigen binding portions, thereof.

[0106] By "subject" is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline.

[0107] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0108] As used herein, the terms "treat.” treating.” "treatment.” and the like refer to reducing, abating, diminishing, or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0109] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0110] By “vector’ is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell. In one embodiment, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

[0111] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0112] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0113] 13

[0114] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0115] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0116] BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIGs. 1A-1D illustrate the identification of high-affinity HLA-A*02:01 TP53-R175H T cell receptors (TCRs) using a yeast display system. FIG. 1 A shows readouts of fluorescence-activated cell sorting (FACS) readouts of T cells that were transduced with T cell receptors (TCRs) generated during two cycles of affinity maturation to determine a TCR's specificity for a p53 peptide comprising an R175H mutation. FIG. IB is a graph showing monomer binding of representative clones generated during the first two cycles of affinity maturation. FIG. 1C shows FACS flow readouts of cells that were generated during a third cycle of affinity maturation and express a TCR that binds a p53 peptide comprising an R175H mutation. FIG. ID is a graph showing monomer binding of representative clones generated during the third cycle of affinity maturation.

[0118] FIGs. 2A-2D show that T cell engagers (TCEs) generated during affinity maturation demonstrate robust and specific binding to HLA-A*02:01 TP53-R175H and human T cells. FIG. 2A is a graph showing dose-dependent binding of T cell engagers to immobilized HLA- A*02 p53-R175H. FIG. 2B is a graph showing dose-dependent binding ofT cell engagers (TCEs) to immobilized HLA-A*02 wild type p53. FIG. 2C is a graph showing the percentage of five TCEs that are specific for p53 R175FI-FILA-A*02:01 complexes that also bind to T cells. FIG. 2D is a graph showing increased dose-dependent T cell binding of each of the five TCEs. as quantified by mean fluorescence intensity (MFI). ‘‘Secondary Only"’ denotes the baseline level of background signal as determined by an experimental run without TCEs.

[0119] FIG. 3 is a graph showing that aT cell engager of the present disclosure (i.e., TCE1) mediates robust and specific T cell activation by HLA-A*02 TP53-R175H, but not wild ty pe p53 protein or an off- target protein (MART-1), as measured by IFNg expression.

[0120] FIGs. 4A-4D demonstrate that a T cell engager of the present disclosure (i.e., TCE1) demonstrates potent and specific killing of endogenous HLA-A*02:01 TP53-R175H- expressing tumor cells. FIG. 4A is a graph showing HLA-A*02 expression by four different cell lines (KMS26, KLE, SK-MEL-5, and SKBR3). FIG. 4B is a graph showing expression of p53 peptides comprising an R175H mutation in KMS26. KLE. SK-MEL-5, and SKBR3 cells. FIG. 4C is a graph showing TCEl-mediated tumor cell killing of KMS26 tumor cells. FIG. 4D is a graph showing TCEl-mediated tumor cell killing of KLE and SKBR3 cells.

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[0122] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0123] FIG. 5 is a graph showing an X-Scan analysis that suggests no potential off-targets for TCE1 in the human proteome.

[0124] FIGs. 6A and 6B demonstrate that TCE1 does not mediate T cell activation towards normal human tissue. FIG. 6 A is a graph showing HLA-A*02 expression in normal human epidermal keratinocytes (NHEK), human bronchial epithelial cells (HBEpC). human aortic endothethial cells (HAoEC), and T2 cells. FIG. 6B is a graph showing TCE 1 -mediated T cell activation (as measured by IFNg expression) in normal human tissues and T2 cells. The bars are presented in groupings of ten, and are ordered, from left to right, for the first five bars: TCE 1 at concentrations of 0 nM, 0. 1 nM, 1 nM, 10 nM, and 100 nM each with the addition of 1 pM TP53-R175H Peptide, and for the second five bars TCE 1 at concentrations of 0 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM without TP53-R175H Peptide.

[0125] FIG. 7 is a schematic showing the structure of an exemplary TCE of the present disclosure. The exemplary TCE includes an anti-CD3 binding domain (e.g., a single chain anti-CD3 antibody) and a single chain T cell receptor (scTv).

[0126] FIGs. 8A-8C provide graphs showing that TCEs designed to target TP53-R175H demonstrated in vitro killing of antigen-positive tumor cell lines, while showing no effect against antigen-negative tumor cell lines. FIG. 8A is a graph showing that 4 different TCEs induced tumor cell killing when added to HLA-A*02+, TP53-R175H+ human myeloma (KMS26) cells. FIG. 8B is a graph showing that 4 different TCEs induced tumor cell killing when added to HLA-A*02+, TP53-R175H+ human adenocarcinoma (KLE) cells. FIG. 8C is a graph showing that none of the 4 different TCEs induced tumor cell killing when added to HLA-A*02+, TP53-R175H- human melanoma (SK-MEL-5) cells.

[0127] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0128] Provided and described herein are biological products, compositions and methods for treating cancer, including tumors and neoplasias.

[0129] “T cell engagers (TCEs)” are bispecific peptides that simultaneously bind a tumor cell and a T cell. TCEs are relatively easy to manufacture and dose relative to adoptive cell therapies. TCEs effectively form a bridge, connection, or synapse between the cells, thereby facilitating T cell-mediated cytotoxicity, e.g., against a tumor, cancer, or neoplasia cell.

[0130] The present disclosure provides T cell engagers (TCEs) that can be used to treat or prevent cancer. In some embodiments, a TCE comprises a T cell receptor (TCR) or an antigen binding fragment thereof that is capable of binding a peptide-MHC complex

[0131] 15

[0132] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 presented on the surface of a cancer, tumor, or neoplasia cell. TCEs generally have higher binding affinities for the peptide MHC complex and lower dissociation rates than TCRs, which allows for a more stable, longer-lasting binding of the TCE to the tumor cell.

[0133] TCEs also comprise a binding moiety that specifically binds a surface protein on a T cell. The binding moiety may specifically bind a protein involved in T cell function. For example, the binding moiety (e.g., antibody or antigen binding fragment thereof) in some embodiments specifically binds to a CD3 protein that is a TCR co-receptor involved in activating a cytotoxic T cell. Without being bound by theory, a TCE bound to a tumor cell can effectively activate any cytotoxic T cell by binding to CD3 expressed on the T cell surface, including those T cells having TCRs that do not bind the same peptide-MHC complex that the TCE binds, through its interaction with CD3 on the T cell surface. Activated cytotoxic T cells release lytic agents (e.g., perforins and granzymes) that cause death of the cancer, tumor, or neoplasia cell. In some embodiments, activation of a T cell allows for proliferation of the T cell.

[0134] In some embodiments, the TCE comprises one or more domains that increases the half- life of the TCE. In some embodiments, the domain that increases the half-life of the TCE is an Fc domain. A fragment cry stall izable domain or region (Fc domain or region) constitutes the “tail” region of an antibody molecule that interacts with cell surface Fc receptors and some proteins of the complement system. The Fc domain allows binding proteins such as antibodies and the like to activate the immune system, for example, through binding to Fc receptors present on the surface of cells. The Fc domain can be fused at the N- or C-terminus of the TCE or via a linker to one or more intervening amino acids.

[0135] In some embodiments, TCEs comprise one or more antigen-binding domains of a full length antibody. In some embodiments, the TCE comprises a heavy chain variable (Vn) region and a light chain variable (VL) region of an antibody that specifically binds a protein involved in T cell function (e.g., CD3 polypeptide). In some embodiments, the TCE comprises an antibody that lacks a constant region. In some embodiments, the TCE comprises a TCR domain and an antibody domain or an antigen binding fragment thereof wherein the TCR domain and the antibody or antigen binding fragment thereof are arranged in single chain form. In some embodiments, the entire TCE is designed as a single chain. In some embodiments, the TCE comprises a tumor targeting binding domain (e.g., TCR that binds a high-affinity HLA-A*02:01 TP53-R175H complex and an anti-CD3 antibody or antigen binding fragment).

[0136] 16

[0137] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0138] Tumor Suppressor Protein p53 (TP53)

[0139] The tumor suppressor protein p53 (TP53) plays a pivotal role in preventing tumor formation by inducing cell cycle arrest and apoptosis in response to DNA damage. However, TP53 mutations are prevalent across various cancer types, and these mutations not only result in loss of tumor suppressive functions but also confer gain-of-function properties that drive oncogenesis. Among these mutations, HLA-A*02:01 TP53-R175H represents the largest TP53 population with >34,000 cases per year in the US and EU across all solid tumor indications, with no therapies explicitly targeting this mutation in the clinic.

[0140] The practice of the aspects and embodiments described herein employs, unless otherw ise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well -within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology ” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel. 1987); “PCR: The Polymerase Chain Reaction”, (Mullis. 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides as described herein, and, as such, may be considered in making and practicing the described aspects and embodiments. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0141] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the products, compositions and methods as described and are not intended to limit the scope of the aspects and embodiments described herein.

[0142] EXAMPLES

[0143] Example 1: Engineered T cell engagers (TCEs) targeting HLA-A*02:01 TP53 R175H for cancer immunotherapy

[0144] Bispecific T cell engagers were engineered to simultaneously engage T cells via CD3 and target TP53-R175H-expressing cancer cells by an affinity-matured specific TCR.

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[0147] Affinity maturation was performed on an HLA-A*02 TP53-R175H-speciftc TCR isolated from a healthy donor using an internally developed yeast display platform (FIGs. 1A-1C).

[0148] 3 cycles of CDR diversification using a structure-guided approach, with 6 rounds of selection of increasing stringency per cycle, enabled the identification of TCR clones exhibiting significantly increased on-target binding. Cycle 1 screened 64 clones, resulting in the confirmation of 28 unique binders. Cycle 2 screened 232 clones, resulting in the confirmation of 23 unique binders. Cycle 3 screened 192 clones, resulting in the confirmation of 22 unique binders.

[0149] Identified clones demonstrated specificity for the TP53 cognate peptide, comprising an R175H mutation over the wild type peptide presented in an HLA-A*02 context (FIG. ID). Select clones were formatted as T cell engagers (TCEs) for further characterization.

[0150] Example 2: T cell engagers demonstrate robust and specific binding to HLA-A*02:01 TP53-R175H and human T cells

[0151] 5 TCEs of the present disclosure (TCEs 1-5) and a TCE produced by using a TCR that underwent only 2 cycles of CDR diversification (Cycle 2 TCE) were tested against immobilized HLA-A*02 TP53-R175H and HLA-A*02 TP53-WT complexes (FIGs. 2A-2B). TCEs were also tested for binding to T cells (FIGs. 2C-2D).

[0152] Dose-dependent binding to immobilized HLA-A*02 TP53-R175H. but not to wild type TP53 (TP53-WT), suggested that affinity-matured TCRs retain their binding specificity when formatted as T cell engagers (TCE). TCEs 1-5 demonstrated more robust binding to HLA- A*02 TP53-R175H but similar binding to HLA-A*02 TP53-WT relative to the Cycle 2 TCE.

[0153] T cell engagers also demonstrated robust binding to human T cells in a dose-dependent manner and is not impacted by the different TCR binders.

[0154] Example 3: T cell engagers mediate robust and specific T cell activation by HLA-A*02 TP53-R175H

[0155] TCE 1 was selected for further testing of T cell activation. T2 cells pulsed with 1 pM peptide were co-cultured with human pan T cells (E:T ratio of 1 : 1) in the presence of a TP53- R175H specific T cell engager (FIG. 3). IFNg was quantified by Meso Scale Discovery (MSD) assay 16 hours post-co-culture.

[0156] The results showed that TCE 1 mediates specific T cell activation in a dose-dependent manner by the TP53-R175H peptide but not by the irrelevant HLA-A*02-restricted MART-1

[0157] 18

[0158] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 peptide. Minimal T cell activation was observed in the context of HLA-A*02 TP53-WT peptide.

[0159] Example 4: T cell engagers demonstrate potent and specific killing of endogenous HLA- A*02:01 TP53-R175H-expressing tumor cells

[0160] Tumor cells were co-cultured with human pan T cells at an E:T ratio of 10: 1 in the presence of TCE 1. Tumor cell killing was quantified by luciferase 72 hours post-co-culture and normalized to a no T cell engager control condition (FIGs. 4C-4D). HLA-A*02 expression of the tumor lines was confirmed by flow cytometry (FIG. 4A) and TP53-R175H expression was quantified by western blot (FIG. 4B).

[0161] The results demonstrated that TCEs of the present disclosure redirect T cells to mediate specific tumor cell killing against HLA-A*02 TP53-R175H-expressing tumor cells in a dose-dependent manner. The TCEs were able to direct T cell mediated tumor cell killing even against the SK-BR-3 cell line that has notably low expression of HLA-A*02 (FIG. 4D). TCEs did not direct killing of tumor cells not expressing TP53 R175H (SK-MEL-5) (FIG. 4D)

[0162] Example 5: X-Scan analysis suggests no potential off-targets for the T cell engager in the human proteome

[0163] X-Scan analysis was conducted with TCE 1 and T cells to test for off-target activation of T cells by TCE 1 (FIG. 5).

[0164] T2 cells pulsed with 1 pM of a panel of positional scanning peptides containing a substitution of every possible amino acid at each position of the cognate p53 R175H peptide and were incubated with human pan T cells at an E:T ratio of 1: 1 in the presence of 10 nM of the T cell engager. IFNg secretion was quantified by MSD 16 hours post-co-culture. Black boxes indicated the presence of parent residue. Secreted IFNy levels were determined to identify cross-reactivity to promiscuous positions. Potentially antigenic peptides were then matched against the human proteome to predict potential in vivo off-targets using ScanProsite (prosite.expasy.org / scanprosite / ). Peptides that elicited a response of greater than 10% of the cognate peptide signal were considered positive in this assay.

[0165] No potential off-targets were identified in the human proteome suggesting a favorable tolerability profile of the T cell engager.

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[0168] Example 6: T cell engager does not mediate T cell activation towards human normal tissue

[0169] Normal tissue or T2 cells pulsed with or without 1 pM TP53-R175H peptide were incubated with human pan T cells at an E:T ratio of 1 : 1 in the presence of TCE 1. IFNg secretion was quantified by MSD 16 hours post-co-culture (FIG. 6B). HLA-A*02 expression was confirmed by flow cytometry (FIG. 6A).

[0170] Co-culture of normal tissue with human pan T cells in the presence of the T cell engager did not lead to T cell activation, as measured by IFNg secretion, suggesting a favorable tolerability profile.

[0171] Example 7: T cell engagers demonstrated HLA-A*02 TP53-R175H Specific Tumor Killing

[0172] TCEs w ere engineered using a novel single chain structure (FIG. 7).

[0173] Affinity’ maturation was performed on an HLA-A*02 TP53-R175H-specific TCR isolated from a healthy donor using a yeast display platform. The resulting TCRs were engineered as single chain TCRs.

[0174] The single chain TCRs were further engineered to increase tertiary structure stability7. The stabilization process involved random mutagenesis.

[0175] Following stabilization, four TCRs were selected, and fused to a single chain peptide comprising the VH and VL domains of an anti-CD3 antibody to produce four TCEs. Sequences of the four TCEs are found in Table 1 below. Sequences of components of the TCEs are found in Table 2 below-.

[0176] Tumor cells were co-cultured with human pan T cells at an E:T ratio of 10: 1 in the presence of each of the four TCEs. Each of the four TCEs demonstrated the ability to direct killing of human myeloma cells (KMS26) expressing HLA-A*02 and TP53-R175H (FIG. 8A) and human adenocarcinoma cells (KLE) expressing HLA-A*02 and TP53-R175H (FIG. 8B). Each of the four TCEs were specific for TP53-R175H. None of the four TCEs directed killing of cells expressing wild-type TP53 (FIG. 8C). These results demonstrated that the four TCEs directed HLA-A*02 TP53-R175H specific killing of tumor cells.

[0177] Table 1: TCE Sequences

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[0179] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0180] GDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG

[0181] DRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD

[0182] YTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSDSGVTQTPKHL

[0183] IAATGQRVTLRCSPRSGDLSVYWYRQSPDQGLQFLIQYYNGEERGIGNILERFS

[0184] AQQFPDLHSELNISSLELGDSALYFCASGDGIIEQYFGPGTRLTVTEDLKNGGG

[0185] GSGGGGSGGGGSGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWY

[0186] RQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCA

[0187] VSNSNSRAYQLIFGKGTKLSVIPNGGGGSDYKDDDDKHHHHHH

[0188] 21

[0189] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025

[0190] Table 2: Sequences of TCE Components

[0191] ^DLKN is the amino acid sequence of the first five amino acids of the TCRP constant region.

[0192] 22

[0193] ACTIVE 716175404v1 Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025 detection tag to enable assays.

[0194] 3Affinity tag for purification.

[0195] The data demonstrated successful affinity maturation of a donor TCR, resulting in enhanced recognition of the HLA-A*02:01 TP53-R175H peptide complexes with high specificity. After conversion of the affinity matured TCR to a TCE, tumor co-culture assays revealed potent T cell-mediated killing of mutant TP53-R175H-expressing cancer cells by the engager, accompanied by robust T cell activation characterized by cytokine production. Importantly, the engager exhibited favorable tolerability profiles, demonstrated by minimal off-target activation via X-scan and with minimal T cell activation towards normal tissue.

[0196] These findings demonstrated the promising activity and tolerability profile of a novel T cell engager targeting HLA-A*02 TP53-R175H for cancer immunotherapy.

[0197] Other Embodiments

[0198] From the foregoing description, it will be apparent that variations and modifications may be made to the aspects and embodiment described herein for adoption to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0199] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any’ single embodiment or in combination with any other embodiments or portions thereof.

[0200] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference herein.

[0201] 23

[0202] ACTIVE 716175404v1

Claims

Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025What is claimed is:1 . AT cell engager (TCE) comprising a T cell binding domain or an antigen binding fragment thereof and a T cell receptor (TCR) component that specifically binds to a tumor suppressor protein 53 (TP53) polypeptide comprising a mutation presented by a major histocompatibility complex (MHC) molecule binding domain.

2. The TCE of claim 1, wherein the TP53 mutation is TP53 R175EI.

3. The TCE of claim 1 or 2. wherein the MHC molecule comprises human leukocyte antigen (HL A) serotype A2.

4. The TCE of claim 3, wherein the HLA serotype is HLA-A*02:01.

5. The TCE of any one of claims 1-4, wherein the TCR component is derived from an affinity-matured TCR.

6. The TCE of any one of claims 1-5, wherein the T cell binding domain comprises a CD3 binding domain.

7. The TCE of claim 6, wherein the CD3 binding domain comprises an antigen binding domain of an anti-CD3 antibody.

8. The TCE of any one of claims 1-7, wherein the TCE further comprises a half-life extension moiety.

9. The TCE of claim 8, wherein the half-life extension moiety is an Fc domain.

10. A method of eliciting an immune response against a tumor cell in a subject, the method comprising administering to the subject the TCE of any one of claims 1-9.

11. The method of claim 10, wherein the tumor cell is a solid tumor cell.

12. The method of claim 10 or 11, wherein the tumor cell expresses a tumor associated antigen.

13. The method of any one of claims 10-12, wherein the tumor cell expresses an oncogenic driver mutation.24ACTIVE 716175404v1Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 202514. The method of claim 10, wherein the oncogenic driver mutation is a mutation in TP53.

15. The method of claim 14, wherein the TP53 mutation is R175H.

16. The method of any one of claims 10-15, wherein the tumor cell expresses HLA serotype A2.

17. The method of claim 16, wherein the tumor cell expresses HLA serotype HLA- A*02:01.

18. A method of activating a T cell, the method comprising contacting the T cell with the TCE of any one of claims 1-10.

19. AT cell engager (TCE) comprising a CD3 binding domain or an antigen binding fragment thereof, and a T cell receptor (TCR) component, wherein the T cell receptor component specifically binds a tumor suppressor protein 53 (TP53) polypeptide or fragment thereof comprising an R175H mutation presented by a major histocompatibility complex (MHC) molecule.

20. The TCE of claim 19, wherein the TCE comprises a single polypeptide chain comprising the TCR component linked to the CD3 binding domain or antigen binding fragment thereof.

21. The TCE of claim 19 or 20, wherein the CD3 binding domain is an anti-CD3 antibody or antigen-binding fragment thereof.

22. The TCE of claim 21, wherein the CD3 binding domain comprises or consists of a heavy chain variable (Vn) region and a light chain variable (VL) region of an anti-CD3 antibody.

23. The TCE of claim 21, wherein the CD3 binding domain does not include a constant region of an anti-CD3 antibody.

24. The TCE of any one of claims 19-23, wherein the CD3 binding domain comprises a VH region of an anti-CD3 antibody comprising an amino acid sequence having at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK25ACTIVE 716175404v1Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSS.

25. The TCE of any one of claims 19-24, wherein the CD3 binding domain comprises a VL region of an anti-CD3 antibody comprising an amino acid sequence having at least 85% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK.

26. The TCE of any one of claims 19-25, wherein the CD3 binding domain comprises an amino acid sequence having at least 85% sequence identity toEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIR NYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK.

27. The TCE of any one of claims 19-26, wherein the TCE comprises an amino acid sequence having at least 85% sequence identity toEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIR NYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIKGGGGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVY WYRQSPDQGLQFLIQYYNGEERGIGNILERFSAQQFPDLHSELNISSLELGDSALYFC ASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGGGSGGGGSGGGSQKEVEQNSGPLSV PEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIPNGGGGSDYKDDDDK HHHHHH.

28. The TCE of any one of claims 19-26, wherein the TCE comprises an amino acid sequence having at least 85% sequence identity toEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIR26ACTIVE 716175404v1Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 2025NYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIKGGGGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVY WYRQSPDQGLQFLIQYYNGEERGIGNILERFSAQQFPDLHSELNISSLELGDSALYFC ASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGGGSGGGGSGGGSQKEVEQNSGPLSV PEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIPNGGGGSDYKDDDDK HHHHHH.

29. The TCE of any one of claims 19-26, wherein the TCE comprises an amino acid sequence having at least 85% sequence identity toEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIR NYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIKGGGGSDSGVTQTPKHLIAATGQRVTLRCTPRSGDLSV YWYQQSPDQGLQFLIQYYNGEERGIGNIPERFSAQQFPDLHSELNLSSLELGDSALYF CASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGGGSGGGGSGGGSQKEVEQNSGPLS VPEGANASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKDDGRFTAQLNKAS QYVSLLIRDSQPSDSATYLCAVSNSNSRAYQLIFGKGTKLSVIPNGGGGSDYKDDDD KHHHHHH.

30. The TCE of any one of claims 19-26, wherein the TCE comprises an amino acid sequence having at least 85% sequence identity toEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFD VWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIR NYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSDSGVTQTPKHLIAATGQRVTLRCSPRSGDLSVY WYKQSPDQGLQFLIQYYNGEERGIGNIPERFSAQQFPDLHSELNLSSLELGDSASYFC ASGDGIIEQYFGPGTRLTVTEDLKNGGGGSGGGGSGGGGSGGGSQKEVEQNSGPLSV PEGAISSLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQY VSLL1RDSQPSDSATYLCAVSNSNSRAYQLTFGKGTKLSV1PYGGGGSDYKDDDDKH HHHHH.27ACTIVE 716175404v1Atorney Docket No.: 218378-040702 / PCT Electronic Deposit Date: November 4. 202531. The TCE of any one of claims 1-9 or claims 19-30, wherein the TCE comprises from N-terminus to C-terminus or from C-terminus to N-terminus, the CD3 binding domain or an antigen binding fragment thereof, and the T cell receptor (TCR) component.

32. A polynucleotide encoding the TCE of any one of claims 1-9 or 19-31.

33. A vector comprising the polynucleotide of claim 32.

34. The vector of claim 33, wherein the vector is a plasmid vector.

35. The vector of claim 33, wherein the vector is a viral vector.

36. The vector of claim 35, wherein the vector is an adeno-associated viral (AAV) vector.

37. A cell comprising the polynucleotide of claim 32 or the vector of any one of claims33-36.

38. A method of eliciting an immune response against a tumor cell in a subject, the method comprising administering to the subject a TCE of any one of claims 1-9 or claims 19- 31, or the vector of any of claims 33-36.

39. A method of activating a T cell, the method comprising contacting the T cell with the TCE of any one of claims 1-9 or claims 19-31.

40. A pharmaceutical composition comprising a TCE of any one of claims 1-5 or claims 19-31, or the vector of any of claims 33-36, and a pharmaceutical excipient.

41. A kit for immunotherapy, the kit comprising the TCE of any one of claims 1-5 or claims 19-31, or the vector of any of claims 33-36.

42. A method of treating a neoplasia in a subject, the method comprising administering to the subject the TCE of any one of claims 1-5 or claims 19-31, or the vector of any of claims 33-36.

43. The method of claim 42, wherein the neoplasia is adenocarcinoma and / or myeloma28ACTIVE 716175404v1

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