Host cells with KRAS binding proteins and knockout of endogenous TCRs and methods of use thereof

Abstract

The present disclosure provides methods and compositions for adoptive T cell therapy, in particular extracellular binding proteins that target KRAS peptides, host cells comprising the binding proteins, and methods of use and production thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the following U.S. provisional patent applications: 63 / 483,231 filed February 3, 2023; 63 / 492,146 filed March 24, 2023; 63 / 496,356 filed April 14, 2023; 63 / 503,403 filed May 19, 2023; and 63 / 545,883 filed October 26, 2023, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Adoptive T cell therapy (ACT) has demonstrated activity in solid tumors but requires further optimization to become more reproducibly effective. T cells engineered with T cell receptors (TCRs) that recognize intracellular oncogenic drivers such as mutant KRAS, the most frequently altered gene in human cancers, have the potential to induce durable responses in patients with solid tumors. Summary of the Invention

[0003] overview There is a need for improved engineered host cells and compositions for adoptive cell therapy for solid tumors. Without wishing to be bound by theory, it is understood that competition between transgenic and endogenous TCRs for the available CD3 pool on T cells, and the possibility of mispairing of transgenic and endogenous TCR chains, can result in reduced surface expression of engineered TCRs and, therefore, reduced sensitivity when engineered TCRs are used in adoptive cell therapy.

[0004] Thus, some aspects of the present disclosure relate to host cells that have an exogenous transgenic mutant KRAS-targeted TCR, and the endogenous TCR is edited to eliminate the expression of the endogenous TCR and mispairing with the transgenic TCR. In some cases, this manipulation involves the use of type VA CRISPR nuclease in parallel with compatible gRNA, resulting in TCR knockout in more than 90% of human primary T cells. In some embodiments, this editing has extremely high specificity for knocking out TCR-related loci, improving the expression of the exogenous TCR and improving the sensitivity of the engineered host cells to therapeutically targeted cells (for example, enhancing the in vitro cytotoxicity of tumor cells that express mutant KRAS G12D peptide).

[0005] In one aspect, the disclosure provides a host cell comprising a heterologous extracellular binding protein, wherein the extracellular binding protein is capable of binding to a peptide:HLA complex, and the peptide comprises a KRAS G12D mutant peptide, and a genomic mutation that reduces expression of endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2).

[0006] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding a heterologous extracellular binding protein inserted into a TRAC, TRBC1, or TRBC2 locus, the extracellular binding protein being capable of binding to a peptide:HLA complex, the peptide comprising a KRAS G12D mutant peptide, and the host cell having reduced expression of TRAC, TRBC1, or TRBC2.

[0007] In another aspect, the present disclosure provides a polynucleotide encoding an extracellular binding protein. The extracellular binding protein may comprise a TCR alpha chain variable (Vα) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 123, 139, 155, 171, 187, 229, 230, 239, 240, 249, 250, 259, 260, 269, and 270; a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 131, 147, 163, 179, 195, 234, 235, 244, 245, 254, 255, 264, 265, 274, and 275; or a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-8, 10-16, 18-24, 26-32, 34-40, 42-48, 50-56, 58-64, 66-72, 74-80, 82-88, 90-96, 98-104, 106-112, 124-130, 132-138, 140-146, 148-154, 156-162, 164-170, 172-178, 180 -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278, comprising a TCRα FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a TCRβ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, comprising an amino acid sequence having at least 80% sequence identity to any one of

[0008] In another aspect, the present disclosure provides a vector comprising the polynucleotide of any one of the above aspects, or embodiments thereof.

[0009] In another aspect, the present disclosure provides a cell, the cell comprising the polynucleotide or vector of any of the above aspects, or embodiments thereof.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition comprising a host cell of any of the above aspects, or embodiments thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0011] In another aspect, the present disclosure provides a method for treating a disease or disorder associated with KRAS G12 mutation in a subject. The method comprises administering to the subject an effective amount of the host cell or pharmaceutical composition of any one of the above aspects or embodiments thereof.

[0012] In any of the above aspects, or embodiments thereof, the peptide:HLA complex comprises an HLA protein encoded by the HLA-A*11 allele. In any of the above aspects, or embodiments thereof, the peptide:HLA complex comprises an HLA protein encoded by the HLA-A*11:01 allele.

[0013] In any of the above aspects, or embodiments thereof, the extracellular binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) region, a TCR beta chain variable (Vβ) region, a T cell receptor (TCR) alpha chain constant (Cα) region, or a T cell receptor (TCR) beta chain constant (Cβ) region.

[0014] In any of the above aspects, or embodiments thereof, the KRAS G12D mutant peptide comprises the amino acid sequence VVVGADGVGK.

[0015] In any of the above aspects, or embodiments thereof, the Vα or Vβ domains are human, humanized, or chimeric.

[0016] In any of the above aspects, or embodiments thereof, the extracellular binding protein is human, humanized, or chimeric.

[0017] In any of the above aspects, or embodiments thereof, the extracellular binding protein comprises a TCR alpha chain variable (Vα) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 123, 139, 155, 171, 187, 229, 230, 239, 240, 249, 250, 259, 260, 269, and 270; a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 131, 147, 163, 179, 195, 234, 235, 244, 245, 254, 255, 264, 265, 274, and 275; or SEQ ID NOs: 2-8, 10-16, 18-24, 26-32, 34-40, 42-48, 50-56, 58-64, 66-72, 74-80, 82-88, 90-96, 98-104, 106-112, 124-130, 132-138, 140-146, 148-154, 156-162, 164-170, 172-178, 180 -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278, comprising a TCRα FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a TCRβ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, comprising an amino acid sequence having at least 80% sequence identity to any one of

[0018] In any of the above aspects, or embodiments thereof, the extracellular binding protein specifically binds to the KRAS G12D mutant peptide.

[0019] In any of the above aspects, or embodiments thereof, the extracellular binding protein is at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold selective for the KRAS G12D mutant peptide over other 10-mer peptides encoded by the genome of the cell.

[0020] In any of the above aspects, or embodiments thereof, the host cell further comprises (i) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or (ii) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain.

[0021] In any of the above aspects, or embodiments thereof, the extracellular binding protein has a β-glucan concentration of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less. or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less log KRAS G12 mutant peptide 10 It has an EC50.

[0022] In any of the above aspects, or embodiments thereof, when the host cell is in the presence of tumor cells expressing the KRAS G12D mutant peptide, CD137 expression on the host cell is elevated compared to (i) CD137 expression by a reference human T cell that does not express the binding protein when the reference human T cell is in the presence of tumor cells; or (ii) CD137 expression by a human T cell that expresses the binding protein when not in the presence of tumor cells or when not in the presence of antigen-presenting cells that express the peptide:HLA complex.

[0023] In any of the above aspects, or embodiments thereof, the genomic mutation that causes or contributes to reduced expression of endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2) comprises an indel in the TRAC, TRBC1, or TRBC2 locus. In any of the above aspects, or embodiments thereof, the genomic mutation that causes or contributes to reduced expression of endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2) is a missense mutation that causes or contributes to reduced function or stability of the T cell receptor alpha or T cell receptor beta polypeptide encoded by the genome of the cell. In any of the above aspects, or embodiments thereof, a genomic mutation that reduces expression of endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBCl), or T cell receptor beta constant 2 (TRBC2) results in premature termination of the T cell receptor alpha or T cell receptor beta polypeptide encoded by endogenous TRAC, TRBCl, or TRBC2. In any of the above aspects, or embodiments thereof, the host cell comprises a genomic mutation that reduces expression of both (i) TRAC; and (ii) TRBCl or TRBC2. In any of the above aspects, or embodiments thereof, the host cell comprises a genomic mutation that reduces expression of TRAC, TRBCl, and TRBC2.

[0024] In any of the above aspects, or embodiments thereof, the host cells comprise immune cells or precursors thereof. In any of the above aspects, or embodiments thereof, the immune cells comprise T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In any of the above aspects, or embodiments thereof, the immune cells comprise T cells, wherein the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - double-negative T cells, γδ T cells, or any combination thereof.

[0025] In any of the above aspects, or embodiments thereof, a polynucleotide encoding a heterologous extracellular binding protein is inserted into the TRAC locus and the host cell has reduced expression of TRAC.

[0026] In any of the above aspects, or embodiments thereof, the host cell further comprises a recombinant protein comprising an IL-7 receptor alpha (IL7RA) intracellular domain and an IL7RA transmembrane domain. In any of the above aspects, or embodiments thereof, the IL7RA intracellular domain has at least 80% sequence identity to SEQ ID NO: 224. In any of the above aspects, or embodiments thereof, the IL7RA transmembrane domain has at least 80% sequence identity to SEQ ID NO: 225.

[0027] In any of the above aspects, or embodiments thereof, the recombinant protein further comprises a CD34 extracellular domain or a CD58 extracellular domain. In any of the above aspects, or embodiments thereof, the CD58 extracellular domain has at least 80% sequence identity to SEQ ID NO: 227. In any of the above aspects, or embodiments thereof, the recombinant protein has at least 80% sequence identity to SEQ ID NO: 223.

[0028] In any of the above aspects, or embodiments thereof, the binding protein is capable of binding to a peptide:HLA complex, and the peptide comprises a KRAS G12 mutant peptide. In any of the above aspects, or embodiments thereof, the KRAS G12 mutant peptide is a KRAS G12D mutant peptide. In any of the above aspects, or embodiments thereof, the KRAS G12D mutant peptide comprises the amino acid sequence VVVGADGVGK.

[0029] In any of the above aspects, or embodiments thereof, the nucleic acid sequence is codon optimized.

[0030] In any of the above aspects, or embodiments thereof, the extracellular binding protein is human, humanized, or chimeric.

[0031] In any of the above aspects, or embodiments thereof, the extracellular binding protein is selective for the KRAS G12D mutant peptide.

[0032] In any of the above aspects, or embodiments thereof, the extracellular binding protein has a β-glucan concentration of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less. or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less log KRAS G12 mutant peptide 10 It has an EC50.

[0033] In any of the above aspects, or embodiments thereof, the extracellular binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 229, 230, 234, 235, 239, 240, 244, 245, 249, 250, 254, 255, 259, 260, 264, 269, 265, 270, 274, and 275.

[0034] In any of the above aspects, or embodiments thereof, the polynucleotide further comprises a promoter. In any of the above aspects, or embodiments thereof, the promoter is the elongation factor-1 alpha (EF-1α) promoter.

[0035] In any of the above aspects, or embodiments thereof, the polynucleotide comprises RNA, DNA, or a combination thereof.

[0036] In any of the above aspects, or embodiments thereof, the vector is a lentiviral vector, a gamma-retroviral vector, or an adeno-associated viral (AAV) vector.

[0037] In any of the above aspects, or embodiments thereof, the pharmaceutical composition comprises both CD4+ cells and CD8+ cells having (i) an extracellular binding protein and (ii) a genomic mutation that reduces expression of endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2).

[0038] In any of the above aspects, or embodiments thereof, the pharmaceutical composition further comprises either or both of (i) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or (ii) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain.

[0039] In any of the above aspects, or embodiments thereof, the composition comprises about a 1:1 ratio of CD4+ T cells to CD8+ T cells.

[0040] In any of the above aspects, or embodiments thereof, the subject is positive for the HLA-A*11 allele.In any of the above aspects, or embodiments thereof, the subject is positive for the HLA-A*11:01 allele.

[0041] In any of the above aspects, or embodiments thereof, the KRAS G12 mutation is a KRAS G12D mutation.

[0042] In any of the above aspects, or embodiments thereof, the disease or disorder comprises cancer. In any of the above aspects, or embodiments thereof, the cancer is a solid cancer. In any of the above aspects, or embodiments thereof, the cancer is a hematological malignancy.

[0043] In any of the above aspects, or embodiments thereof, the disease or disorder is selected from the group consisting of bile duct tumors, cholangiocarcinoma, colon adenocarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC); colorectal cancer; lung cancer, non-small cell lung cancer; biliary tract cancer; endometrial cancer; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, myeloid leukemia, acute myeloid leukemia; myelodysplastic syndrome; lymphoma, non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); cancer of the urinary tract; cancer of the small intestine; breast cancer; melanoma, cutaneous melanoma, anal melanoma, or mucosal melanoma; glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neuroblastoma. fibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; lung squamous cell carcinoma; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

[0044] In any of the above aspects, or embodiments thereof, the subject is determined to carry the KRAS G12D allele prior to the administering step. In any of the above aspects, or embodiments thereof, the subject has been genotyped for an HLA-A allele prior to the administering step.

[0045] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings given below unless otherwise specified.

[0046] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0047] By "agent" is meant a polypeptide, nucleic acid molecule, small compound, or cell that comprises a heterologous polynucleotide. In some embodiments, the cell is an immune cell (e.g., a T cell) that is autologous or heterologous to the subject.

[0048] "Alteration" means a change (increase or decrease) in the expression level, structure, or activity of a gene or polypeptide as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change in expression level, a 25% change, a 40% change, or a 50% or greater change in expression level.

[0049] By "ameliorate" is meant to lessen, inhibit, attenuate, reduce, arrest, or stabilize the onset or progression of a disease.

[0050] As used herein, the term "CD8 coreceptor" or "CD8" generally refers to the cell surface glycoprotein CD8. CD8 exists on the cell surface as either a CD8α subunit-CD8α subunit homodimer or a CD8α subunit-CD8β subunit heterodimer. The CD8 coreceptor supports the function of cytotoxic T cells (CD8+) and functions through signal transduction via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). There are five documented human CD8β chain isoforms (see UniProtKB identifier P10966) and a single documented human CD8α chain isoform (see UniProtKB identifier P01732).

[0051] "CD8 alpha" or "CD8α" refers to a polypeptide having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001759.3 or NP_741969.1, or a fragment thereof that has coreceptor activity for ligand recognition by the T cell receptor when dimerized with another CD8α or CD8β. An exemplary CD8α amino acid sequence is provided below. TIFF2026504480000001.tif50149

[0052] "CD8 beta" or "CD8β" refers to a polypeptide having at least about 85% amino acid sequence identity to NCBI Accession Nos. XP_054200532.1, NP_757362.1, NP_742099.1, NP_742100.1, NP_004922.1, or NP_001171571.1, or a fragment thereof that has coreceptor activity for ligand recognition by the T cell receptor when dimerized with CD8α. An exemplary CD8β amino acid sequence is provided below. TIFF2026504480000002.tif172159

[0053] In this disclosure, "comprises," "comprising," "containing," "having," and the like may have the meaning given to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning given to them in U.S. patent law, and the terms are open-ended, allowing for the existence of more than what is recited, but excluding aspects of the prior art, so long as the basic or novel characteristics of what is recited are not altered by the existence of more than what is recited.

[0054] "Complementary" means that it can pair to form a double-stranded nucleic acid molecule or a portion thereof. In one embodiment, the antisense molecule is mostly complementary to the target sequence. Complementarity does not need to be perfect, and can contain mismatches at one, two, three, or more nucleotides.

[0055] By "corresponding" is meant that a strand of the double-stranded inhibitory nucleic acid molecule contains at least a fragment of the double-stranded gene such that it can bind to a complementary strand of the gene.

[0056] The terms "complementarity determining region" and "CDR" generally refer to a sequence of amino acids in an immunoglobulin superfamily member (e.g., TCR) variable region that confers antigen specificity or binding affinity and is separated from each other in the primary amino acid sequence by framework regions. Generally, each TCR α chain variable region has three CDRs (αCDR1, αCDR2, αCDR3), and each TCR β chain variable region has three CDRs (βCDR1, βCDR2, βCDR3). In TCRs, CDR3 is considered to be the main CDR responsible for recognizing processed antigens. Generally, CDR1 and CDR2 interact primarily or exclusively with MHC.

[0057] CDR1 and CDR2 are encoded within the variable gene segments of the TCR variable region coding sequence, while CDR3 is encoded by the region spanning the variable segment and connecting segment for Vα, or the region spanning the variable segment, diversity segment, and connecting segment for Vβ. Therefore, when the identity of the variable gene segment of Vα or Vβ is described, the corresponding CDR1 and CDR2 sequences can be inferred, for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3 may be significantly more diverse due to the addition and loss of nucleotides during the recombination process.

[0058] TCR variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), which allows equivalent residue positions to be annotated and allows for comparison of different molecules using, for example, the ANARCI software tool (2016, Bioinformatics 15:298-300). The numbering scheme provides a standardized depiction of framework regions and CDRs in TCR variable domains. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme or method (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, the CDRs of the present disclosure are identified according to the Kabat numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified according to the Chothia numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified according to the EU numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified according to the enhanced Chothia numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified according to the Aho numbering scheme or method.

[0059] By "reduce" is meant a decrease of at least about 5% compared to a reference level. The decrease can be 5%, 10%, 15%, 20%, 25%, or 50%, or even as much as 75%, 85%, 95%, or more, and any intervening percentage.

[0060] "Detecting" refers to determining the presence, absence, or amount of the analyte to be detected.

[0061] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In embodiments, the disease is cancer.

[0062] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The level of expression of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in 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 daughter cells during cell division. Because its expression is restricted to the transfected cell, gene expression 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 selective advantage to the transfected cell. Such a selective advantage may be resistance to a certain toxin presented to the cell.

[0063] "Effective amount" means the amount required to improve the symptoms of a disease compared to an untreated patient. The effective amount of the active compound used to practice the present invention for the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0064] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, the portion containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0065] The terms "isolated," "purified," or "biologically pure" refer to material that is free, to varying degrees, from components that normally accompany it as found in its native state. "Isolated" refers to some degree of separation from the original source or surroundings. "Purified" refers to a degree of separation that goes beyond isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so 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 invention 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. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. For proteins that may be subject to modifications, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be purified separately.

[0066] "Isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that does not contain the genes adjacent to the genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term includes recombinant DNA that is incorporated into, for example, a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0067] By "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, a 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, preparations are at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the invention. Isolated polypeptides of the invention can 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, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0068] By "marker" is meant any analyte or clinical condition having an alteration that can be characterized as described herein. In embodiments, the analyte is a protein or polynucleotide marker, e.g., a marker of differentiation, having increased or decreased expression associated with a disease state or cellular condition.

[0069] As used herein, "immune cells" generally refer to any cell of the immune system. In embodiments, immune cells originate from hematopoietic stem cells in the bone marrow and give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, regulatory T cells, natural killer cells, natural killer T cells, and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of the T cell.

[0070] As used herein, a "heterologous" or "foreign" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule, or a portion of a nucleic acid molecule, that is not native to a host cell but can be homologous to a nucleic acid molecule or portion thereof derived from the host cell. The source of a heterologous or foreign nucleic acid molecule, construct, or sequence can be from a different genus or species. In certain embodiments, a heterologous or foreign nucleic acid molecule (i.e., not endogenous or native) is added to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, etc., where the added molecule can be integrated into the host genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can exist in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, polypeptide, or other activity encoded by a foreign nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity. Furthermore, a cell that contains a "modified" or "heterologous" polynucleotide or binding protein includes the progeny of that cell, whether the progeny themselves have been transduced, transfected, or otherwise manipulated or altered.

[0071] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).

[0072] "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 an appropriate molecule (e.g., a transcription activator protein) is bound to the second polynucleotide. Thus, the described components are in a relationship that allows them to function in their intended manner. For example, placing a coding sequence under the regulatory control of a promoter means positioning the coding sequence such that expression of the coding sequence is controlled by the promoter.

[0073] As used herein, the term "promoter" refers to a DNA sequence that directs the expression (transcription) of a gene. A promoter can direct the transcription of a gene in a prokaryotic or eukaryotic organism. A promoter can be "inducible," which initiates transcription in response to an inducer, or, in contrast, "constitutive," in which an inducer does not regulate the rate of transcription. A promoter can be regulated in a tissue-specific or tissue-preferential manner so that it is only active in transcribing an operably linked coding region in a specific tissue type or types.

[0074] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0075] "Reference" means a standard or control condition.

[0076] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset 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 a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally 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 therebetween.

[0077] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the invention.

[0078] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, more preferably 80% or 85%, and 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0079] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of 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 typically 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, closely related sequences are identified using the 'e' (e.g., 'e' ... -3 ~e -100 The BLAST program may be used, with a probability score between .

[0080] By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0081] Ranges provided herein are understood to be shorthand for all of the values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0082] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms. Although not excluded, it will be understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0083] As used herein, "T cells" or "T lymphocytes" generally refer to immune cells that mature in the thymus and produce a T cell receptor (TCR). T cells can be naive ("TN"; not exposed to antigen; compared to TCM (described herein), they have increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased or no expression of CD45RO), memory T cells (TM) (antigen-experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced and cytotoxic). TM can be further divided into subsets of central memory T cells (TCM; expressing CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM; expressing CD45RO and reduced expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TEs) are antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have reduced expression of CD62L, CCR7, and CD28 compared to TCMs, and are positive for granzymes and perforin. Helper T cells (THs) are CD4+ cells that affect the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress adaptive immune responses, and which of these two functions is induced depends on the presence of other cells and signals. T cells can be collected using suitable techniques, and various subpopulations or combinations thereof can be enriched or depleted by suitable techniques, such as affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+CD25+ (Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8+CD28-, and Qa-1-restricted T cells.

[0084] As used herein, "T cell receptor" (TCR) generally refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail that can specifically bind to an antigenic peptide bound to an MHC receptor; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997. TCRs can be found on the surface of cells or in soluble form and generally comprise a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively), or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively).

[0085] The term "variable region" or "variable domain" generally refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR α or β chain (or γ and δ chains for γδ TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains of the α and β chains of native TCRs (Vα and Vβ, respectively) generally have similar structures, with each domain containing four generally conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); the Vβ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind to a specific antigen can be isolated using the Vα or Vβ domain from the antigen-binding TCR to screen a library of complementary Vα or Vβ domains, respectively.

[0086] As used herein, "vector" generally refers to a macromolecule or a group of macromolecules that contains or associates with a polynucleotide and can be used to mediate the delivery of the polynucleotide to cells.Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles.Vectors generally contain genetic elements, such as regulatory elements, that are functionally linked to genes to facilitate the expression of the genes in the target.

[0087] The present disclosure includes variants of any of the enzymes described herein that have one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R chain length for each other. Such conservatively substituted variants can include variants that have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the sequences listed in Table 1. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can include sequences with substitutions of key binding residues of the polypeptide or polynucleotide such that the activity is not destroyed.

[0088] Conservative substitution tables providing functionally similar amino acids are available from various references (see, for example, Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co.; 2nd edition (December 1993)). These tables are divided into eight groups: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) each contain amino acids that are conservative substitutions for one another. [Brief explanation of the drawings]

[0089] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0090] [Figure 1A]Figures 1A and 1B show TCR activation in a preliminary screening of KRAS-G12D TCRs. Plots of the percentage of GFP-positive cells as a function of peptide concentration and the resulting EC50 charts are shown. Peptide dose-dependent responses for each TCR were assessed by analyzing GFP expression after overnight culture with A11 target cells pulsed with decreasing concentrations of peptide as indicated. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism®. TCR091 showed the highest affinity in the assay. [Figure 1B] See legend to Figure 1A. [Figure 2] Figure 1 shows a comparison of the cytotoxic activity of wild-type TRAC / TRBC T cells and TRAC / TRBC double knockout (dKO) T cells transduced to express a KRAS-G12D-specific TCR. Red-fluorescent Hpaf-II cells, a KRAS-G12D-expressing tumor cell line transduced to express HLA-A11, were cocultured with TCR32-transduced CD8+ T cells harboring wild-type TRAC and TRBC loci, or TCR32-transduced TRAC / TRBC double knockout (dKO) CD8+ T cells, as indicated, at a 3:1 effector:target cell ratio. Red fluorescence was measured by live-cell imaging using an IncuCyte S3 microscope and software package. Total red object integrated intensity is plotted over time as a measure of tumor cell volume. [Figure 3A] Figures 3A, 3B, 3C, 3D, and 3E show a comparison of T cell activation in wild-type TRAC / TRBC T cells (upper panels) and TRAC / TRBC dKO T cells (lower panels) transduced to express a KRAS-G12D-specific TCR. Plots of the percentage of CD137-positive cells assessed via FACS versus peptide concentration are shown. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4-1]Figures 4A-4J show characterization of the cytotoxic activity of wild-type TRAC / TRBC T cells and TRAC / TRBC dKO T cells transduced to express a KRAS-G12D-specific TCR. Plots of total red object integrated intensity are shown as a measure of tumor cell volume over time, as measured by live-cell imaging using an IncuCyte S3 microscope and software package, as described for Figure 2. Figure 4A provides data for T cells harboring a wild-type TRAC / TRBC locus incubated with Hpaf-II target cells at a 3:1 effector-to-target ratio. Figure 4B provides data for T cells harboring a TRAC / TRBC double knockout incubated with Hpaf-II target cells at a 3:1 effector-to-target ratio. Figure 4C provides data for T cells harboring a wild-type TRAC / TRBC locus (upper panel) or a TRAC / TRBC double knockout (lower panel) incubated with HuCCT1 target cells at a 3:1 effector-to-target ratio. Figure 4D provides data for T cells with a wild-type TRAC / TRBC locus (upper panel) or a TRAC / TRBC double knockout (lower panel) incubated with HuCCT1 target cells at an effector-to-target ratio of 3:1. Figure 4E provides data for T cells with a wild-type TRAC / TRBC locus or a TRAC / TRBC double knockout (see legend) incubated with HuCCT1 target cells at an effector-to-target ratio of 6:1. Figure 4F provides data for T cells with a wild-type TRAC / TRBC locus or a TRAC / TRBC double knockout incubated with HuCCT1 target cells. Figure 4G provides data for T cells with a wild-type TRAC / TRBC locus or a TRAC / TRBC double knockout (see legend) incubated with Hpaf-II target cells at an effector-to-target ratio of 3:1.Figure 4H and Figure 4I provide data for T cells from two donors harboring wild-type TRAC / TRBC loci (upper panel) or a TRAC / TRBC double knockout (lower panel) incubated with Hpaf-II target cells at an effector-to-target ratio of 3:1. Figure 4J provides data for T cells harboring wild-type TRAC / TRBC loci (upper panel) or a TRAC / TRBC double knockout (lower panel) incubated with Panc-1 target cells at an effector-to-target ratio of 3:1. Figure 4K summarizes the efficiency of wild-type TRAC / TRBC T cells and TRAC / TRBC dKO T cells transduced to express KRAS-G12D-specific TCRs for killing Hpaf-II, HuCCT1, and Panc-1 target cells. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 4-5] See description of Figure 4-1. [Figure 4-6] See description of Figure 4-1. [Figure 4-7] See description of Figure 4-1. [Figure 4-8] See description of Figure 4-1. [Figure 4-9] See description of Figure 4-1. [Figure 5-1] FIG. 5A shows the scheme of X-scan analysis to identify potential off-target TCR interaction partners. [Figure 5-2] Figures 5B-5E show charts depicting potentially cross-reactive peptides identified from ScanProsite searches for TCR91 (Figure 5B), TCR2 (Figure 5C), TCR4 (Figure 5D), and TCR5 (Figure 5E). [Figure 5-3] See description of Figure 5-2. [Figure 6]10 demonstrates that TCR91 exhibits superior specificity for the KRAS G12D peptide compared to the RASL11B peptide. Plots of the percentage of CD137 positive cells are shown alongside the derived EC50 and Cmax tables. [Figure 7] Figure 1 shows that dKO enhances TCR sensitivity compared to single knockout (sKO). A plot of the percentage of CD137 positive cells is shown alongside a table of derived EC50 and Cmax values. [Figure 8] Figure 1 shows a comparison of Hpaf-II tumor cell killing by sKO and dKO cells expressing a KRAS G12D-recognizing TCR. Cytotoxic activity was assessed and plots of red fluorescence measured over time were generated by live cell imaging. [Figure 9] Figure 1 shows enhanced killing of Panc-1 tumor cells by T cells harboring the TRAC / TRBC dKO and expressing a KRAS G12D-recognizing TCR. Data were generated using the same protocol as described for Figure 8, but with Panc-1 tumor cells instead of HpafII tumor cells. [Figure 10] Figures 10A and 10B show tetramer analysis of TCR91-transduced primary CD4+ / CD8+ T cells carrying wild-type TRAC / TRBC loci, TRBC single knockout (sKO), or TRAC / TRBC dKO. Cell surface expression of the correctly paired G12D TCR and its functionality were assessed using fluorescent dye-labeled tetramers of MHC-peptide complexes. Figure 10A shows a bar graph of the percentage of tetramer binding for each condition, and Figure 10B shows the mean fluorescence intensity (MFI) values. [Figure 11] Stimulation of KRASG12D-specific TCR-T cells with the G12D 10-mer is shown. Plots of the percentage of CD137-positive cells versus peptide concentration are shown for all cells (upper panel) or transduced cells (lower panel). TCR91-transduced TRAC / TRBC dKO cells showed increased activation compared to WT and TRBC sKO cells. [Figure 12]Figure 2 shows the analysis of T cell cytotoxic activity of TCR91-transduced primary CD4+ / CD8+ T cells harboring a wild-type TRAC / TRBC locus, a TRBC single knockout (sKO), or a TRAC / TRBC dKO. Cytotoxic activity was assessed using the IncuCyte assay described for Figure 2. [Figure 13] Figures 13A and 13B show the results of experiments in which primary CD4+ and CD8+ T cells were transduced with a KRAS-G12D-specific T cell receptor (TCR-91) at a transduction efficiency of 35% (Figure 13A) or 70% (Figure 13B) with or without knockout of TRAC and TRBC. Plots of total red object integrated intensity (a measure of tumor cell volume) over time are shown, obtained by live cell imaging using an IncuCyte S3 microscope and software package as described for Figure 2. The results demonstrate that TRAC / TRBC dKO cells are more effective at controlling HpafII tumor cells than TRAC / TRBC wild-type cells. [Figure 14] Figures 14A and 14B show the results of experiments in which primary CD4+ and CD8+ T cells were transduced with KRAS-G12D-specific T cell receptor 91, with or without TRAC and TRBC knockout, at a transduction efficiency of 35% (Figure 14A) or 70% (Figure 14B). Cytotoxic activity against Panc-1 tumor cells was assessed at an effector:target cell ratio of 10:1 using an IncuCyte assay. The results demonstrate that TRAC / TRBC dKO cells are more effective in controlling Panc-1 tumor cells than TRAC / TRBC wild-type cells. [Figure 15]Figures 15A and 15B show tetramer analysis of TCR91-transduced primary CD4+ / CD8+ T cells harboring a wild-type TRAC / TRBC locus, a TRAC single knockout (sKO), or a TRAC / TRBC dKO. Cell surface expression of the correctly paired G12D TCR and its functionality were assessed using fluorochrome-labeled tetramers of MHC-peptide complexes. Bar graphs are shown for each condition: the percentage of cells positive for tetramer binding (Figure 15A) and the level of tetramer binding to cells measured by mean fluorescence intensity (MFI) (Figure 15B). TRAC / TRBC dKO cells showed greater tetramer binding compared to WT or TRAC sKO cells. Figure 15C provides a representative scatter plot showing the cell surface expression of the correctly paired G12D TCR and its binding to fluorochrome-labeled tetramers of MHC-peptide complexes. Primary CD4+ and CD8+ T cells were transduced with TCR with or without TRAC / TRBC dKO. [Figure 16] Figure 16A shows tumor volume over time for NSG mice implanted subcutaneously with CL40 colon adenocarcinoma cells and treated with 10x10^6 T cells (control T cells with knockout of endogenous TRAC and TRBC, or T cells expressing a KRAS G12D-specific TCR disclosed herein with co-expression of the CD8 co-receptor (CD8αβ) and knockout of endogenous TRAC and TRBC) administered intravenously 9 days after implantation. Figure 16B shows survival curves for NSG mice implanted subcutaneously with CL40 colon adenocarcinoma cells and treated with 10x10^6 T cells (control T cells with knockout of endogenous TRAC and TRBC, or T cells expressing a KRAS G12D-specific TCR disclosed herein with co-expression of the CD8 co-receptor (CD8αβ) and knockout of endogenous TRAC and TRBC) administered intravenously 9 days after implantation. [Figure 17-1]Figure 17A shows enhanced STAT5 phosphorylation in engineered cells with the addition of a chimeric fusion protein comprising an IL-7 receptor intracellular signaling domain. "IL2" indicates a control condition in which cells were treated with recombinant IL2 to induce STAT5 phosphorylation. Figure 17B shows proliferation of T cells engineered to comprise a KRAS G12D-specific TCR disclosed herein in response to tumor cells. Data comparing engineered T cells with or without the addition of a chimeric fusion protein comprising an IL-7 receptor intracellular signaling domain is shown. Figure 17C shows tumor cell killing by T cells engineered to comprise a KRAS G12D-specific TCR disclosed herein with or without the addition of a chimeric fusion protein comprising an IL-7 receptor intracellular signaling domain. [Figure 17-2] Figure 17D shows tumor volume over time for mice subcutaneously implanted with HuCCT1 cells and treated with 1x10^7 T cells (control T cells with knockout of endogenous TRAC and TRBC, or T cells expressing a KRAS G12D-specific TCR disclosed herein with co-expression of the CD8 co-receptor (CD8αβ) and knockout of endogenous TRAC and TRBC, with (+ILR) or without (-ILR) a chimeric fusion protein comprising the IL-7 receptor intracellular signaling domain) administered intravenously 9 days after implantation. [Figure 18A] 1 is a chart showing the efficiency of non-viral knock-in of the G12D KRAS TCR disclosed herein in primary CD4+ and CD8+ T cells. [Figure 18B] 1 shows killing of Panc1 tumor cells by T cells engineered to contain a KRAS G12D-specific TCR as disclosed herein via lentiviral transduction (LV) or non-viral knock-in technology (KI). [Figure 18C] 1 shows killing of HuCCT1 tumor cells by T cells engineered to contain a KRAS G12D-specific TCR as disclosed herein via lentiviral transduction (LVV) or non-viral knock-in technology (KI). [Figure 19] Figure 19A shows the percentage of CD4 / CD8 T cells expressing CD3 on the cell surface after targeting the TRAC and TRBC loci with nuclease MG29-1. Activity was comparable to that of the CRISPR / Cas9 system. Figure 19B shows the percentage of indel frequency at the TRAC and TRBC loci after MG29-1-mediated gene editing. UTD = untransduced. [Figure 20] Indel activity was assessed across 590 predicted potential off-targets (OTs) selected across TRAC and TRBC gRNAs with up to six mismatches. High on-target editing efficiency was observed, but no off-target activity was detected above background, exceeding the assay's quantitation limit (0.05%). [Figure 21] Oligo-capture analysis was performed in primary T cells to further evaluate the specificity of these nuclease / gRNA combinations. [Figure 22-1]Figure 22A shows the frequency of translocation events between the TRAC and TRBC loci in edited primary T cells, as detected by dPCR. Figure 22B shows the frequency of translocation events between the TRAC and TRBC loci, as detected by karyotyping. Figures 22C and 22D show a comparison of activation, measured by the percentage of 2A+ T cells expressing CD137, of T cells engineered to express a KRAS-G12D-specific TCR and bearing either a wild-type TRAC / TRBC locus (Figure 22C) or a TRAC sKO or TRAC / TRBC dKO (Figure 22D), after stimulation with the cognate peptide recognized by the TCR. Figure 22E shows a comparison of activation, measured by the percentage of 2A+ T cells expressing CD137, of T cells bearing either a TRAC sKO or a TRAC / TRBC dKO after coculture with A11 antigen-presenting cells. Figures 22F-22M show a comparison of activation, as measured by the percentage of 2A+ T cells expressing CD137, of T cells engineered to express a KRAS-G12D-specific TCR and harboring either a wild-type TRAC / TRBC locus or a TRAC sKO or TRAC / TRBC dKO, after coculture with A11-expressing cancer cell lines. Figure 22F shows data for T cells engineered with pGE106, pGE116, pGE107, or pGE129 constructs after coculture with HPAF-II cells. Figure 22G shows data for T cells engineered with pGE106, pGE116, pGE107, or pGE129 constructs after coculture with HuCCT1 cells. Figure 22H shows data for T cells modified with pGE106, pGE116, pGE107, or pGE129 constructs after co-culture with Panc1 cells. Figure 221 shows data for T cells modified with pGE106, pGE116, pGE107, or pGE129 constructs after co-culture with CL40 cells. Figure 22J shows data for T cells modified with pGE106, pGE114, or pGE129 constructs after co-culture with HPAF cells.Figure 22K shows data for T cells modified with pGE106, pGE114, or pGE129 constructs after coculture with HuCCT1 cells. Figure 22L shows data for T cells modified with pGE106, pGE114, or pGE129 constructs after coculture with Panc1 cells. Figure 22M shows data for T cells modified with pGE106, pGE114, or pGE129 constructs after coculture with CL40 cells. Figures 22N-22T show a comparison of in vitro proliferation of T cells modified via electroporation-mediated knock-in or LVV-mediated transduction to express a KRAS-G12D-specific TCR and harboring either a wild-type TRAC / TRBC locus or a TRAC sKO or TRAC / TRBC dKO, after coculture with A11-expressing cancer cell lines. Figure 22N shows data for cells engineered to express either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, after coculture with HuCCT1 cells at an effector-to-target ratio of 3:1. Figure 22O shows data for cells engineered to express either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, after coculture with Panc1 cells at an effector-to-target ratio of 3:1. Figure 22P shows data for cells engineered to express either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, after coculture with HPAF-II cells at an effector-to-target ratio of 1:1. Figure 22Q shows data for cells engineered to express either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, after co-culture with CL40 cells at an effector-to-target ratio of 1:1. Figure 22R shows data for T cells engineered with pGE106, pGE116, pGE107, or pGE129 constructs after co-culture with HPAF cells at an effector-to-target ratio of 1:1. Figure 22S shows data for T cells engineered with pGE106, pGE116, pGE107, or pGE129 constructs after co-culture with HuCCT1 cells at an effector-to-target ratio of 6:1.Figure 22T shows data for T cells engineered with pGE106, pGE116, pGE107, or pGE129 constructs after coculture with Panc1 cells at an effector-to-target ratio of 6: 1. Figure 22U shows a comparison of the cytotoxic activity of T cells engineered to express either TCR2, CD34-IL7R, and CD8α / β, or TCR2, CD58-IL7R, and CD8α / β, after coculture with HuCCT1 cells at an effector-to-target ratio of 10: 1. [Figure 22-2] See description of Figure 22-1. [Figure 22-3] See description of Figure 22-1. [Figure 22-4] See description of Figure 22-1. [Figure 22-5] See description of Figure 22-1. [Figure 22-6] See description of Figure 22-1. [Figure 22-7] See description of Figure 22-1. [Figure 22-8] See description of Figure 22-1. [Figure 22-9] See description of Figure 22-1. [Figure 22-10] See description of Figure 22-1. [Figure 22-11] See description of Figure 22-1. [Figure 23] This figure shows that T cells (G12D TCR-T cells) engineered by non-viral targeted knock-in (KI) of the TCR alpha constant chain (TRAC) locus to express a multicistronic cassette containing 1) a high-affinity TCR specific for the KRAS G12D mutation, 2) a CD8αβ co-receptor, and 3) a chimeric cytokine receptor bind KRAS G12D peptide with high functional avidity and exhibit robust cytotoxicity in vitro. G12D TCR-T TCR T cells specifically bound KRAS G12D peptide at subnanomolar concentrations (left) and exhibited robust cytotoxicity against HuCCT1 tumor cells (endogenous KRAS G12D and HLA-A*11-01) even upon tumor cell rechallenge. [Figure 24]

[0023] Figure 1 provides graphs showing that engineered T cells exhibit robust tumor cell control in vivo. Engineered CD4 / CD8 TCR T cells were administered intravenously to NSG mice after subcutaneous inoculation of HuCCT1 tumor cells (left) or CL40 tumor cells (right). Treatment with G12D TCR-T cells resulted in durable and robust responses in a mouse xenograft model. [Figure 25]

[0023] Figure 1 provides heat maps and graphs showing that G12D TCR-T cells exhibit a low risk of cross-reactivity. Engineered TCR T cells were incubated with a library of X-scan peptides, and TCR T cell activation was measured to reveal the recognition motif of the KRAS G12D TCR. The recognition motif was used to scan for matches against human peptides. None of the potentially cross-reactive peptides activated the TCR T cells, even at supraphysiological concentrations of 500 nM, demonstrating the high specificity of the KRAS G12D TCR. [Figure 26] Figures 26A-26B provide graphs showing that gene editing reagents exhibit high specificity. To evaluate off-target activity for gene editing reagents, potential off-target sites were identified using in silico prediction (Figure 26A) and oligo-capture analysis (Figure 26B) performed on primary T cells. Each of these potential off-targets was assayed for insertions and deletions in G12D TCR-T cells using a targeted sequencing assay. G12D TCR-T cells exhibited high on-target activity, while none of the potential off-targets exhibited significant off-target activity, confirming the high specificity of the GE reagents used to produce G12D TCR-T cells. [Figure 27]A graph is provided showing that non-viral KI achieves high efficiency of transgene integration. Non-viral knock-in can achieve over 40% transgene integration efficiency in T cells derived from healthy donors. The process performed similarly at research scale and a 10-fold scale-up version. The engineered T cells were expanded and robustly achieved cell doses sufficient for clinical application, demonstrating manufacturability. [Figure 28] This figure provides a graph showing that KI-engineered T cells from patient donors exhibit robust cytotoxicity. T cells from patient donors and healthy donors were engineered with non-viral KI and evaluated for KI efficiency, growth kinetics, and functionality using in vitro cytotoxicity assays. Patient TCR T cells functioned similarly to healthy donor TCR T cells. [Figure 29]

[0023] Figure 1 provides a schematic diagram and graphs showing that lentiviral delivery is limited by cargo size. The schematic diagram shows the transgene construct used in Example 14. The graphs show that viral titers decrease with increasing transgene size (left) and that transduction efficiency decreases with increasing transgene size (right). [Figure 30A] Figures 30A-30B provide schematics and graphs showing that non-viral KI can achieve high transgene integration frequencies even with large transgenes. Figure 30A shows a schematic of a transgene inserted into the endogenous TRAC gene via CRISPR / Cas-driven homology-directed repair, allowing for larger cargo capacity. [Figure 30B] Figures 30A-30B provide a schematic diagram and graphs showing that non-viral KI can achieve high transgene integration frequencies even with large transgenes. Figure 30B shows a graph showing that the optimized KI process can achieve approximately 50% KI efficiency to generate sufficient numbers of engineered TCR T cells to meet clinical doses. The KI process does not result in asymmetric T cell populations. [Figure 31]

[0023] Figure 1 provides a graph showing improved tetramer binding by KI cells. KI-engineered TCR T cells showed improved binding to KRAS G12D tetramers even with fewer transgene vector copies (VCN) per cell, indicating higher TCR expression driven by the EF-1a promoter. [Figure 32] 1 provides a graph showing increased avidity for KI cells. KI-engineered TCR T cells bound KRAS G12D peptide with higher functional avidity than LVV cells, consistent with increased TCR expression. [Figure 33] 1 provides a graph showing that KI cells exhibit excellent activity in vivo. KI cells exhibit excellent tumor control after a single intravenous administration of 5 million CD4 / CD8 TCR-T cells 10 days after subcutaneous inoculation of HuCCT1 tumor cells in NSG mice. [Figure 34] A schematic diagram of EF-1α and promoterless constructs for KI is provided. Non-viral KIs offer the flexibility of utilizing endogenous or exogenous promoters such as EF-1α. Utilizing the endogenous TRAC promoter to drive the transgene allows for controlled expression of the native TCR. [Figure 35] 1 provides a graph showing reduced transgene expression with the TRAC promoter. The EF-1a promoter drives higher levels of TCR, CD8ab, and ILR compared to the endogenous TRAC promoter. [Figure 36] 1 provides a graph showing lower functional avidity with the TRAC promoter. KI-engineered TCR T cells utilizing the EF-1a promoter bind the KRAS G12D peptide with higher functional avidity than cells utilizing the TRAC promoter. [Figure 37] 1 provides a graph showing lower in vivo activity with the TRAC promoter. KI cells utilizing the EF-1α promoter exhibit superior tumor control after a single intravenous administration of 5 million CD4 / CD8 TCR-T cells 10 days after subcutaneous inoculation of HuCCT1 tumor cells in NSG mice. [Figure 38] A schematic diagram of the study in Example 15 is provided. The schematic diagram contains the following abbreviations: 2L+ = second-line plus; BOIN12 = Bayesian optimal interval phase I / II; CRC = colorectal cancer; DL = dose level; DLT = dose-limiting toxicity; EOS = end of study; HLA = human leukocyte antigen; ICF = informed consent form; KRAS = Kirsten rat sarcoma virus; NSCLC = non-small cell lung cancer; OBD = optimal biological dose; PD = progressive disease; PDAC = pancreatic ductal adenocarcinoma; PTFD = post-treatment loss to follow-up; SMC = safety monitoring committee; TCR = T-cell receptor. DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description While various aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the aspects of the invention described herein may be employed.

[0092] The practice of some methods disclosed herein employs immunological, biochemical, chemical, molecular biology, microbiology, cell biology, genomics, and recombinant DNA techniques, unless otherwise indicated. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) (incorporated herein by reference in its entirety); Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); Methods in Enzymology (Academic Press, Inc.) series; PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0093] Host Cells or Compositions In some aspects, the present disclosure provides a host cell comprising an extracellular binding protein, wherein the binding protein can bind to a peptide:HLA complex, and the peptide comprises a KRAS G12 mutant peptide. In some embodiments, the peptide:HLA complex comprises an HLA-A*11 allele. In some embodiments, the peptide:HLA complex comprises an HLA-A*11:01 allele. In some embodiments, the peptide:HLA complex comprises an HLA allele that binds or is predicted to bind to a KRAS mutant peptide (e.g., a G12 mutant peptide such as G12D) with an affinity suitable for presentation and TCR activation, for example, a binding affinity or KD of at most 1000 nM, at most 750 nM, at most 500 nM, at most 250 nM, at most 100 nM, at most 50 nM, or at most 10 nM.

[0094] In some cases, the host cell comprises an immune cell or a precursor thereof. In some cases, the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In some cases, the immune cell is a T cell, and the T cell comprises a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a CD4+CD8+ double positive T cell, a γδ T cell, or any combination thereof. In some cases, the host cell further comprises a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α (CD8α) chain or a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β (CD8β) chain. In some cases, when the host cell is in the presence of tumor cells that express the KRAS G12D mutant peptide, CD137 expression on the host cell is elevated compared to (i) CD137 expression by a reference human T cell that does not express the binding protein when the reference human T cell is in the presence of tumor cells; or (ii) CD137 expression by a human T cell that expresses the binding protein when not in the presence of tumor cells or when not in the presence of antigen-presenting cells that express the peptide:HLA complex.

[0095] host cell The present disclosure provides host cells (e.g., engineered immune cells) and populations thereof that contain, encode, and / or are capable of expressing the extracellular binding proteins disclosed herein.

[0096] The host cell can be a peripheral blood mononuclear cell (PBMC). The host cell can be a lymphoid cell. The host cell can be a lymphocyte. The host cell can be a T cell. The host cell can be a B cell. The host cell can be a natural killer (NK) cell. The host cell can be a natural killer T (NKT) cell. The host cell can be a mammalian cell. The host cell can be a human cell.

[0097] The host cell can be a primary cell. The host cell can be an immortalized cell. The host cell can be a cell line. The host cell can be differentiated from a stem cell, such as an induced pluripotent stem cell (iPSC), an embryonic stem cell, a hematopoietic stem cell (HSC), etc.

[0098] In some cases, the host cells are "off-the-shelf" cells engineered from immune cell lines, such as T cell lines or NK cell lines (e.g., NK-92, or, for example, NK-YS, KHYS-1, NKL, NKG, SNK-6, or IMC-1). Such host cells are readily available and can be formulated for direct administration to a subject in need thereof.

[0099] The host cell can be an αβ T cell. The host cell can be a γδ T cell. In some embodiments, the host cell comprises a disruption or deletion of one or more endogenous TCR-encoding genes, such as TRAC, TRB (e.g., TRBC1 and / or TRBC2), TRG, and / or TRD. In some embodiments, the host cell comprises a disruption or deletion of the variable region of one or more endogenous TCR-encoding genes, such as a disruption or deletion in TRAC, TRB, TRG, and / or TRD. In some embodiments, the host cell comprises a disruption or deletion of the constant region of one or more endogenous TCR-encoding genes.

[0100] Extracellular binding proteins The extracellular binding protein can comprise a TCR or a portion thereof. In some embodiments, the extracellular binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) region, a TCR beta chain variable (Vβ) region, a T cell receptor (TCR) alpha chain constant (Cα) region, and / or a T cell receptor (TCR) beta chain constant (Cβ) region. The extracellular binding protein can also comprise a TCR alpha chain variable (Vα) domain; a TCR beta chain variable (Vβ) domain; a TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a combination thereof.

[0101] The extracellular binding protein can include a component of a TCR signaling complex, for example, the extracellular domain, transmembrane domain, and / or cytoplasmic domain of a TCR signaling complex, such as a human TCR signaling complex.

[0102] The extracellular binding protein can comprise (i) the extracellular domain of a TCR alpha, beta, gamma, or delta chain constant region; (ii) the transmembrane domain of a TCR alpha, beta, gamma, or delta chain; and / or (iii) the cytoplasmic domain of a TCR alpha, beta, gamma, or delta chain. The extracellular binding protein can comprise a full-length or substantially full-length TCR alpha, beta, gamma, and / or delta chain.

[0103] In some cases, the extracellular binding protein comprises a TCR alpha chain variable (Vα) domain; a TCR beta chain variable (Vβ) domain; a TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprising a sequence having at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any of the TCR Va, Vβ, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences set forth in Table 1.

[0104] The extracellular binding proteins disclosed herein, the TCR alpha chain variable (Vα) domain; the TCR beta chain variable (Vβ) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least It can comprise, consist essentially of, or consist of an amino acid sequence having 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity.

[0105] The extracellular binding proteins disclosed herein, TCR alpha chain variable (Vα) domain; TCR beta chain variable (Vβ) domain; TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, can be selected from the TCRs disclosed in Table 1. for any one of the Vα, Vβ, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences, is at most about 70%, at most about 71%, at most about 72%, at most about 73%, at most about 74%, at most about 75%, at most about 76%, at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about It can comprise, consist essentially of, or consist of an amino acid sequence having about 88%, at most about 89%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 95.5%, at most about 96%, at most about 96.5%, at most about 97%, at most about 97.5%, at most about 98%, at most about 98.5%, at most about 99%, or at most about 99.5% sequence identity or similarity.

[0106] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Vα) domain; the TCR beta chain variable (Vβ) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 1109, about 1111, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138, about 139%, about 140%, about 141%, Comprises, consists essentially of, or consists of an amino acid sequence having about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity.

[0107] The degree of sequence identity between two sequences can be determined by comparing two sequences using computer programs designed for this purpose, such as global alignment algorithms or local alignment algorithms.Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ or other suitable methods or algorithms.Global alignment algorithms, such as Needleman and Wunsch algorithms, can be used to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps.Default settings can be used.

[0108] To generate a similarity score for two amino acid sequences, a scoring matrix can be used that assigns positive scores to some non-identical amino acids (e.g., amino acids with similar physicochemical properties and / or amino acids that show frequent substitutions in orthologs, homologs, or paralogs). Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0109] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Vα) domain; the TCR beta chain variable (Vβ) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises, consists essentially of, or consists of the amino acid sequence of any one of the TCR Vα, Vβ, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences disclosed in Table 1.

[0110] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions compared to any one of the sequences disclosed in Table 1.

[0111] For example, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region can comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions compared to any one of the sequences disclosed in Table 1.

[0112] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having an insertion of at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acids compared to any one of the sequences disclosed in Table 1.

[0113] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertion compared to any one of the sequences disclosed in Table 1.

[0114] The one or more insertions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be contiguous, non-contiguous, or a combination thereof.

[0115] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid deletions compared to any one of the sequences disclosed in Table 1.

[0116] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid deletions compared to any one of the sequences disclosed in Table 1.

[0117] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions compared to any one of the sequences disclosed in Table 1.

[0118] The one or more deletions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0119] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions compared to any one of the sequences disclosed in Table 1.

[0120] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions compared to any one of the sequences disclosed in Table 1.

[0121] In some embodiments, the extracellular binding protein, the TCR alpha chain variable (Valpha) domain; the TCR beta chain variable (Vbeta) domain; the TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or the TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions compared to any one of the sequences disclosed in Table 1.

[0122] The one or more substitutions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the one or more substitutions are conservative. In some embodiments, the one or more substitutions are non-conservative.

[0123] In some cases, the extracellular binding protein comprises a human, humanized, or chimeric TCR alpha chain; a TCR beta chain; a TCR alpha chain variable (Valpha) domain; a TCR beta chain variable (Vbeta) domain; a TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region.

[0124] In some cases, the KRAS G12 mutant peptide is a KRAS G12D mutant peptide. In some cases, the KRAS G12 mutant peptide comprises the amino acid sequence VVVGADGVGK. In some cases, the extracellular binding protein is selective for the KRAS G12D mutant peptide, for example, specifically, selectively, or preferentially binds to the KRAS G12D mutant peptide. In some cases, the extracellular binding protein is at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 10,000-fold selective for the KRAS G12D mutant peptide relative to other 10-mer peptides, for example, corresponding wild-type peptides, or peptides encoded by the genome of cells (for example, predicted to bind to different KRAS G12-specific TCRs or exhibit off-target binding to the extracellular binding protein).

[0125] In some cases, the extracellular binding protein is about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less, KRAS It has a log10EC50 against the G12 mutant peptide.

[0126] In some embodiments, the host cells disclosed herein react to a target antigen of an extracellular binding protein (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex) at a concentration of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 The host cell may comprise an extracellular binding protein (e.g., a TCR) that binds with an EC50 (e.g., a peptide dose that achieves half-maximal activation of a T cell population) of less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, or less than about 100 pM. The host cell may comprise, for example, a modification (e.g., a genomic mutation) that results in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2, or a combination thereof. The extracellular binding protein may be a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein.

[0127] In some embodiments, the host cells disclosed herein bind to a target antigen of an extracellular binding protein (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex) at least about 100 mM, at least about 10 mM, at least about 1 mM, at least about 500 μM, at least about 100 μM, at least about 50 μM, at least about 10 μM, at least about 5 μM, at least about 4 μM, at least about 3 μM, at least about 2 μM, at least about 1 μM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, at least about 60 nM, at least about 50 nM, at least about 40 The host cell may comprise an extracellular binding protein (e.g., a TCR) that binds with an EC50 (e.g., a peptide dose that achieves half-maximal activation of a T cell population) of at least about 30 nM, at least about 20 nM, at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 500 pM, or at least about 100 pM. The host cell may comprise, for example, a modification (e.g., a genomic mutation) that results in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2, or a combination thereof. The extracellular binding protein may be a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein.

[0128] In some embodiments, the extracellular binding protein (e.g., TCR) binds to a target (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex) at less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 The antibody binds with a KD of less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, or less than about 100 pM.

[0129] Modifications to reduce expression of endogenous TCR genes The host cell can further comprise one or more modifications (e.g., genomic mutations) that cause or contribute to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus. Reduced expression of TRAC, TRBC1, and / or TRBC2 can, for example, reduce mispairing between a transgenic TCR chain (e.g., an anti-KRAS G12D TCR disclosed herein) introduced into the host cell and the endogenous TCR chain, improve functional expression of the transgenic TCR, and improve signaling and functionality of the TCR complex by freeing up the available pool of CD3 protein to bind to the transgenic TCR rather than the endogenous TCR chain.

[0130] In some embodiments, the modification can promote enhanced tumor cell killing in vitro, ex vivo, or in vivo by the engineered immune cells relative to comparable control cells lacking the modification, hi some embodiments, the modification can promote enhanced sensitivity to a given (e.g., low) density of a target antigen (e.g., a KRAS G12 mutant peptide) compared to corresponding control cells lacking the modification.

[0131] In some embodiments, the genomic mutation causing or contributing to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus comprises an indel in the TRAC, TRBC1, or TRBC2 locus. In some embodiments, the genomic mutation causing or contributing to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus is a missense mutation and may result in reduced function or stability of the T cell receptor alpha or T cell receptor beta polypeptide encoded in the genome of the host cell. In some embodiments, the genomic mutation causing or contributing to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus also results in premature termination of T cell receptor alpha or T cell receptor beta polypeptide translated from the genomic mRNA of the cell. In some cases, the host cell contains a genomic mutation that causes or contributes to reduced expression of both (i) TRAC; and (ii) TRBC1 or TRBC2. In some cases, the host cell contains a genomic mutation that causes or contributes to reduced expression of TRAC, TRBC1, and TRBC2. The genomic mutation can be or can include an insertion, for example, of an expression cassette. The genomic mutation can be or can include a deletion. The genomic mutation can be or can include a substitution.

[0132] In some embodiments, the modification includes, for example, a deletion of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the endogenous TRAC, TRBC1, or TRBC2 open reading frame.

[0133] In some embodiments, the modification comprises knocking down the expression of TRAC, TRBC1, or TRBC2, for example, using shRNA or siRNA. In some embodiments, the modification comprises genome disruption. In some embodiments, the modification comprises, for example, the insertion of a transposon or a premature stop codon.

[0134] In some embodiments, expression of endogenous TRAC, TRBC1, and / or TRBC2 is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least In some embodiments, the expression of TRAC, TRBC1, and / or TRBC2 is reduced by at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 5000-fold. In some embodiments, the expression of TRAC, TRBC1, and / or TRBC2 is eliminated or substantially eliminated. In some embodiments, the expression of TRAC, TRBC1, and / or TRBC2 is reduced to below the detection limit. The reduction in the expression of TRAC, TRBC1, and / or TRBC2 can be determined, for example, by flow cytometry assay (e.g., the percentage of positive cells or mean fluorescence intensity in a population of interest).

[0135] In some embodiments, the reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 is found in at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% of the host cells in the population. In some cases, the reduced expression is found by genome sequencing.

[0136] In some embodiments, a population of host cells disclosed herein that includes one or more modifications (e.g., genomic mutations) that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 has at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 90-fold, at least 1 ... fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 5000-fold increased killing of target cells. Target cell killing can be, for example, as measured by an in vitro cytotoxicity assay. The host cell can comprise an extracellular binding protein (e.g., a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex).

[0137] In some embodiments, a population of host cells disclosed herein that include one or more modifications (e.g., genomic mutations) that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 has at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 90-fold, at least 10-fold, at least 10-fold, at least 10-fold, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10 ... The host cell exhibits at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 5000-fold increased activation in response to the target cell. The host cell can comprise an extracellular binding protein (e.g., a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex). Activation can be, for example, as measured by assays to measure expression of activation markers (e.g., CD137, CD69, Granzyme B, CD107a, IFN-γ, TNF-α, IL-12, cytokines, interleukins, interferons) upon exposure to target cells expressing or presenting the target antigen.

[0138] In some embodiments, a population of host cells disclosed herein that includes one or more modifications (e.g., genomic mutations) that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 has at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 90-fold, at least 1 ... The binding avidity of the extracellular binding protein to the target antigen is increased by 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 5000-fold. The host cell can comprise an extracellular binding protein (e.g., a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein) that binds to the target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex). The increase in avidity can be as measured, for example, by assays to measure expression of activation markers (e.g., CD137, CD69, Granzyme B, CD107a, IFN-γ, TNF-α, IL-12, cytokines, interleukins, interferons) upon exposure to target cells expressing or presenting the target antigen, and / or by assays to measure the EC50 (e.g., the peptide dose that achieves half-maximal activation of a T cell population).

[0139] In some embodiments, a population of host cells disclosed herein that contain one or more modifications (e.g., genomic mutations) that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibit at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold increased binding of an extracellular binding protein to a target antigen compared to a population of control cells (e.g., cells without reduced expression of TRAC, TRBC1, and / or TRBC2). The host cells can comprise an extracellular binding protein (e.g., a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex). The increase in binding can be, for example, as measured by an assay involving staining with peptide-HLA multimers (eg, tetramers or pentamers).

[0140] In some embodiments, a population of host cells disclosed herein that includes one or more modifications (e.g., genomic mutations) that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 500-fold increased expression of extracellular binding proteins (e.g., cell surface expression) compared to a population of control cells (e.g., cells in which expression of TRAC, TRBC1, and / or TRBC2 is not reduced). The extracellular binding protein can be a TCR comprising an α chain and a β chain (e.g., having the Vα and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12D mutant peptide, e.g., present in a peptide:HLA complex). Increased expression can be as measured, for example, by an assay comprising staining with a peptide-HLA multimer (e.g., a tetramer or pentamer) specific for the extracellular binding protein.

[0141] In some embodiments, the population of host cells comprises reduced expression of endogenous TRAC compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRBC1 compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRBC2 compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRAC and TRBC1 compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRAC and TRBC2 compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRBC1 and TRBC2 compared to control cells. In some embodiments, the population of host cells comprises reduced expression of endogenous TRAC, TRBC1, and TRBC2 compared to control cells.

[0142] Additional Polypeptides The host cells can be engineered to contain additional modifications in addition to those that cause or contribute to reduced expression of extracellular binding proteins and TRAC, TRBC1, and / or TRBC2.

[0143] The host cell can comprise a transgenic polynucleotide encoding a polypeptide comprising a CD8 co-receptor alpha (CD8α) chain or an extracellular portion thereof, and / or a transgenic polynucleotide encoding a CD8 co-receptor beta (CD8β) chain polypeptide or an extracellular portion thereof. The host cell can be engineered to express a CD8 co-receptor, e.g., a CD8α chain and / or a CD8β chain, as disclosed herein. Illustrative, non-limiting examples of CD8α and CD8β amino acid sequences that can be used include the provided SEQ ID NO: 219, SEQ ID NO: 220, and variants thereof.

[0144] The host cell can contain a transgenic polynucleotide encoding a Fas-41BB fusion protein. The Fas-41BB fusion protein can contain, for example, the extracellular domain of Fas or a FasL-binding fragment thereof and the intracellular signaling domain of 41BB or the signaling domain. The Fas-41BB fusion protein can be useful, for example, for converting a signal initiated by the binding of Fas to its target (e.g., FasL) into a positive (e.g., costimulatory) signal generated by the 4-1BB intracellular signaling domain, thereby improving the anti-cancer immune functionality of the host cell disclosed herein (e.g., increased proliferation, survival in the tumor microenvironment, and metabolism to support T cell activation and memory development). The extracellular component can include all or a portion of the extracellular domain of Fas, or can be shortened to maintain a short spatial distance (e.g., approximately 9 aaa) between the host cell and the interacting partner upon receptor-ligand interaction. The Fas-41BB fusion protein can contain a transmembrane domain, for example, a Fas, 4-1BB, or CD28 transmembrane domain. An illustrative, non-limiting example of a Fas-41BB fusion protein is provided in SEQ ID NO:221.

[0145] The host cell can contain a transgenic polynucleotide encoding a chimeric fusion protein containing an IL7R intracellular signaling domain. The chimeric fusion protein can contain, for example, the intracellular portion of an interleukin-7 receptor A (IL7RA) polypeptide, or a portion or variant thereof that can contribute to IL-7 signaling in the host cell. The chimeric IL7R fusion protein can provide a "signal 3" to, for example, increase STAT5 phosphorylation and host cell functionality, enhance host cell proliferation, increase host cell survival (e.g., in a tumor microenvironment), and / or enhance chemokine receptor expression.

[0146] Interleukin-7 receptor subunit alpha can also be called IL7R-α, IL7RA, IL-7R-alpha, ILRA, interleukin-7 receptor-α, interleukin 7 receptor, cluster of differentiation 127, CD127, or CDW127.

[0147] An IL7R intracellular signaling domain can comprise an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO:224.

[0148] In some embodiments, the IL7R intracellular signaling domain comprises (a) one or more residues of a BOX1 motif corresponding to residues 8-15 (VWPSLPDH) relative to SEQ ID NO: 224 when optimally aligned, or (b) Y185 relative to SEQ ID NO: 224 when optimally aligned. In some embodiments, the IL7R intracellular signaling domain comprises one or more residues of a FERM domain corresponding to residues 1-6 (KKRIKPI) or residues 16-28 (KKTLEHLCKKPRK) relative to SEQ ID NO: 224 when optimally aligned.

[0149] In some embodiments, the chimeric fusion protein comprises an IL7R transmembrane domain. The IL7R transmembrane domain can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 225. In some embodiments, the IL7R transmembrane domain comprises a mutation relative to SEQ ID NO: 225. In some embodiments, the mutation is or comprises an insertion of one or more cysteines and / or one or more prolines into the amino acid sequence of SEQ ID NO: 225. In some embodiments, the mutation enables or facilitates receptor homodimerization. In some embodiments, the mutation comprises an insertion of a cysteine, proline, threonine trimer peptide (CPT) into the transmembrane domain. In some embodiments, the threonine of the CPT insertion is another amino acid other than threonine, and in at least certain cases, the other amino acid is, or is not, a cysteine ​​or a proline.

[0150] In some embodiments, the chimeric fusion protein comprises the transmembrane domain of IL7R, IL2RA, IL2RB, IL2RG, IL14R, IL15R, IL9R, IL21R, CD2, CD40L, CD58, CD80, or SIRPα.

[0151] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising: (i) the extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof capable of binding to a CD28 or CTLA-4 polypeptide; (ii) the extracellular domain of a Cluster of Differentiation 58 (CD58) polypeptide, or a portion or variant thereof capable of binding to a Cluster of Differentiation 2 (CD2) polypeptide; (iii) the extracellular domain of a signal-regulatory protein alpha (SIRPα) polypeptide, or a portion or variant thereof capable of binding to a Cluster of Differentiation 47 (CD47) polypeptide; (iv) the extracellular domain of a Cluster of Differentiation 40L (CD40L) polypeptide, or a portion or variant thereof capable of binding to a CD40 polypeptide; (v) the extracellular domain of a Cluster of Differentiation 2 (CD2) receptor, or a portion or variant thereof capable of binding to a CD58 polypeptide; or (vi) the extracellular domain of a Cluster of Differentiation 34 (CD34) polypeptide.

[0152] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof capable of binding to a CD28 or CTLA-4 polypeptide. In some embodiments, the extracellular domain of CD80 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:226.

[0153] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of a Cluster of Differentiation 58 (CD58) polypeptide, or a portion or variant thereof capable of binding to a CD28 or CTLA-4 polypeptide. In some embodiments, the extracellular domain of CD80 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 227.

[0154] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of CD34. In some embodiments, the extracellular domain of CD34 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:228.

[0155] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or a chimeric IL7R polypeptide disclosed herein) exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold, at least 500-fold, or at least 1000-fold increased proliferation in response to target cells (e.g., presenting a KRAS G12D peptide) compared to a population of control cells (e.g., corresponding cells lacking either the Fas-41BB fusion protein or the chimeric IL7R polypeptide). Proliferation can be as measured, for example, by an in vitro lymphocyte proliferation assay or by measuring host cell number after co-incubation. The host cell can include modifications that result in reduced expression of extracellular binding proteins (e.g., TCRs comprising the Vα and Vβ regions and / or CDRs disclosed herein) and / or endogenous TRAC, TRBC1, and / or TRBC2.

[0156] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or a chimeric IL7R polypeptide disclosed herein) exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold, at least 500-fold, or at least 1000-fold increased target cell killing compared to a population of control cells (e.g., corresponding cells lacking either the Fas-41BB fusion protein or the chimeric IL7R polypeptide). Target cell killing can be as measured, for example, by an in vitro cytotoxicity assay. The host cell can include modifications that result in reduced expression of extracellular binding proteins (e.g., TCRs comprising the Vα and Vβ regions and / or CDRs disclosed herein) and / or endogenous TRAC, TRBC1, and / or TRBC2.

[0157] A nucleic acid encoding a polypeptide disclosed herein (e.g., an extracellular binding protein, a CD8 co-receptor chain or extracellular portion thereof, a Fas-41BB fusion protein, or a chimeric IL7R fusion protein) can encode a signal peptide. In some cases, the polypeptides of the present disclosure include a signal peptide. The signal peptide can be cleaved during processing of the polypeptide; thus, in some cases, the mature polypeptides disclosed herein do not contain a signal peptide.

[0158] A signal peptide at the N-terminus of a protein can be involved in transport of the protein to or through a membrane, transport to a different membranous cellular compartment, or secretion of the protein from the cell. Nucleic acids encoding proteins of the present disclosure can encode a signal peptide that promotes membrane insertion and surface localization of the protein. The signal peptide can be selected for its ability to promote ER processing and cell surface localization of the protein. Any suitable signal peptide can be used. In some cases, the signal peptide can include a G-CSF signal peptide or a CD8α signal peptide. The signal peptide can be about 10 to about 40 amino acids in length. In some cases, the signal peptide is at least about 10, 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or longer. In some cases, the signal peptide is at most about 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or shorter. In some cases, the signal peptide is about 16 to 30 amino acids in length.

[0159] composition In some cases, the present disclosure provides a pharmaceutically acceptable composition comprising a plurality of the host cells described herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some cases, the composition comprises a CD4+ T cell population and / or a CD8+ T cell population having (i) an extracellular binding protein and (ii) one or more genomic mutations that cause or contribute to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, T cell receptor beta constant 2 (TRBC2) locus, or a combination thereof. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition comprises (ii) a CD8+ cell population comprising at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells.

[0160] In some embodiments, the CD4+ cells and / or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain or a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain. In some embodiments, the CD4+ cells and / or CD8+ cells express a CD8 co-receptor, e.g., a CD8α chain and / or a CD8β chain, as disclosed herein.

[0161] In some cases, the composition comprises a CD4+ T cell population and / or a CD8+ T cell population having (i) an extracellular binding protein and (ii) one or more genomic mutations that cause or contribute to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, T cell receptor beta constant 2 (TRBC2) locus, or a combination thereof. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition comprises (ii) a CD8+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells. In some embodiments, the CD4+ cells or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or a resulting polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor beta (CD8β) chain. In some cases, the composition comprises both CD4+ cells and CD8+ cells having (i) an extracellular binding protein and (ii) a genomic mutation that causes or contributes to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus. In some cases, the composition includes both (ii) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain or a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain.

[0162] In some cases, the composition comprises both CD4+ cells and CD8+ cells having either or both of (i) an extracellular binding protein and (ii) a genomic mutation that causes or contributes to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus. In some embodiments, the CD4+ cells or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or a resulting polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain. In some embodiments, the CD4+ cells or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or a resulting polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain. In some embodiments, the composition comprises (i) a CD4+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises (ii) a CD8+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells. In some embodiments, the composition comprises a reduced amount of naive T cells or is substantially free of naive T cells. In some embodiments, the composition comprises about a 1:1 ratio of CD4+ T cells to CD8+ T cells. In some cases, the composition includes both CD4+ cells and CD8+ cells that have either or both of (i) an extracellular binding protein and (ii) a genomic mutation that causes or contributes to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1) locus, or T cell receptor beta constant 2 (TRBC2) locus.In some embodiments, the CD4+ cells or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or a resulting polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain. In some embodiments, the composition comprises (i) a CD4+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises a CD8+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells. In some cases, the composition comprises a reduced amount of naive T cells or is substantially free of naive T cells. In some embodiments, the composition comprises about a 1:1 ratio, about a 1:2 ratio, about a 1:3 ratio, about a 1:4 ratio, about a 1:5 ratio, about a 1:6 ratio, about a 1:7 ratio, about a 1:8 ratio, about a 1:9 ratio, about a 1:10 ratio, about a 2:1 ratio, about a 3:1 ratio, about a 4:1 ratio, about a 5:1 ratio, about a 6:1 ratio, about a 7:1 ratio, about a 8:1 ratio, about a 9:1 ratio, or about a 10:1 ratio of CD4+ T cells to CD8+ T cells.

[0163] In some cases, the carrier or excipient comprises albumin. In some cases, the diluent comprises physiologically normal saline. Suitable excipients can also include water, saline, dextrose, glycerol, etc., and combinations thereof. In some embodiments, the composition comprises a suitable infusion vehicle. The suitable infusion vehicle can be any isotonic vehicle formulation, normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), and can utilize 5% dextrose in water or lactated Ringer's solution. The infusion vehicle can be supplemented with human serum albumin or other human serum components.

[0164] Polynucleotide or Vector In some aspects, the present disclosure provides a polynucleotide comprising an open reading frame encoding an extracellular binding protein (e.g., an extracellular binding protein capable of binding a KRAS mutant peptide). The open reading frame can be operably linked to a promoter (e.g., a heterologous promoter). The extracellular binding protein can include a TCR alpha chain variable (Valpha) domain; a TCR beta chain variable (Vbeta) domain; a TCR alpha FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCR beta FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region disclosed herein, for example, comprising a sequence having at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any of the sequences set forth herein (e.g., in Table 1). The polynucleotide can be codon-optimized. The extracellular binding protein can comprise the amino acid sequence of any one of SEQ ID NOs: 1-112, 118-218, and 229-278.

[0165] In some cases, the heterologous promoter is not a mammalian promoter. In some cases, the heterologous promoter is a constitutive promoter that is not a TCR promoter. In some cases, the heterologous promoter is a viral promoter. In some cases, the heterologous promoter is a mammalian promoter. In some cases, the heterologous promoter is a human promoter. In some cases, the heterologous promoter is a synthetic promoter. In some cases, the heterologous promoter is an inducible promoter. In some cases, the heterologous promoter is a tissue-specific promoter. In some cases, the heterologous promoter is an immune cell-specific promoter.

[0166] In some embodiments, the promoter is a murine stem cell virus (MSCV) promoter. In some embodiments, the polynucleotide is promoterless. In some embodiments, the promoterless polynucleotide is designed to be operably linked to an endogenous promoter at the site of insertion. In some embodiments, the promoter is an elongation factor-1 alpha (EF-1α) promoter. In some embodiments, the promoter has at least about 85% sequence identity to the following exemplary sequences: TIFF2026504480000003.tif164160

[0167] In some embodiments, the binding protein can bind to a peptide:HLA complex, and the peptide comprises a KRAS G12 mutant peptide. In some embodiments, the KRAS peptide is a KRAS G12D mutant peptide. In some embodiments, the KRAS G12D mutant peptide comprises the amino acid sequence VVVGADGVGK. In some embodiments, the extracellular binding protein is human, humanized, or chimeric. In some embodiments, the extracellular binding protein is selective for the KRAS G12D mutant peptide. In some embodiments, the extracellular binding protein is about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less of KRAS It has a log10EC50 against the G12 mutant peptide.

[0168] In some aspects, the present disclosure provides a vector comprising any of the polynucleotides described herein.In some cases, the vector is a viral vector, such as a lentiviral vector, a gamma-retroviral vector, or an adeno-associated viral (AAV) vector.In some embodiments, the vector is a non-viral vector, such as a plasmid, nanoplasmid, minicircle, midge, MIP, or doggybone, lipid-based nanoparticle, liposome, circular polynucleotide (e.g., DNA or RNA), linear polynucleotide (e.g., DNA or RNA), or a combination thereof.

[0169] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A) between the nucleic acid sequence encoding the TCR receptor variable alpha (Vα) region and the nucleic acid sequence encoding the TCR receptor variable beta (Vβ) region. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., T2A) disposed between the TCR receptor and the IL7R fusion protein (e.g., CD58-IL7R or CD34-IL7R). In some embodiments, the IL7R fusion protein comprises the intracellular domain and / or transmembrane domain of the constitutively active IL7R subunit alpha. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A) disposed between the sequence encoding the IL7R fusion protein and the sequence encoding a CD8 co-receptor (e.g., the sequence encoding the CD8 co-receptor alpha chain and the sequence encoding the CD8 co-receptor beta chain). In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., T2A, PTA, E2A, furin peptide, or other self-cleaving peptide) between the sequence encoding the CD8 co-receptor alpha chain and the sequence encoding the CD8 co-receptor beta chain. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a binding protein and a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; and / or a nucleic acid sequence encoding a self-cleaving peptide disposed between the nucleic acid sequence encoding the binding protein and the nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain. In some embodiments, the polynucleotide comprises the following operably linked in-frame: (i)(pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β)-(pnFP); (ii)(pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α)-(pnFP); (iii) (pnBP)-(pnSCP1)-(pnFP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); or (iv)(pnBP)-(pnSCP1)-(pnFP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α) wherein pnCD8α is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnBP is a nucleic acid sequence encoding a binding protein, pnFP is a nucleic acid sequence encoding a fusion protein, and pnSCP1 and pnSCP2 are each independently polynucleotides encoding self-cleaving peptides, the polynucleotides and / or encoded self-cleaving peptides being optionally the same or different. In some embodiments, the self-cleaving peptide is P2A, T2A, E2A, or a furin peptide. In some embodiments, the furin peptide comprises the amino acid sequence RAKR. In some embodiments, the binding protein and the fusion protein are encoded in a single construct or contiguous genome segments. In some embodiments, the binding protein, the fusion protein, and CD8α or CD8β, or both, are encoded in a single construct or contiguous genome segments. In some embodiments, the binding protein and the fusion protein are encoded in a single open reading frame. In some embodiments, the binding protein and the fusion protein are operably linked to a single promoter. In some embodiments, the binding protein and the fusion protein are operably linked to different promoters.

[0170] Treatment method In some aspects, the present disclosure provides a method for treating a disease or disorder associated with KRAS G12 mutation in a subject, comprising administering to the subject an effective amount of any of the host cells or compositions described herein. In some embodiments, the host cells are autologous to the subject. In some embodiments, the host cells are allogeneic to the subject. In some embodiments, the host cells are HLA-matched to the subject, for example, HLA-matched for all typed HLA alleles. In some embodiments, the host cells and the subject can be HLA-typed for HLA-A, HLA-B, HLA-C, and / or HLA-DR alleles. In some embodiments, the host cells and the subject are matched for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 HLA alleles. In some embodiments, the host cells are haploidentical to the subject.

[0171] In some embodiments, the subject is positive for the HLA-A*11 allele. In some embodiments, the subject is positive for the HLA-A*11:01 allele. In some embodiments, the mutation is a KRAS G12D mutation.

[0172] In some embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the disease or disorder comprises pancreatic cancer or cancer of the pancreas, optionally pancreatic ductal adenocarcinoma (PDAC); colon cancer or cancer of the colon; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or cancer of the uterine lining; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia, such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); cancer of the urinary tract; cancer of the small intestine; breast cancer or cancer of the breast; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell carcinoma. Selected from: follicular neoplasm; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; lung squamous cell carcinoma; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

[0173] In some embodiments, the method further comprises, before administering, determining the tumor of the subject for KRAS G12D allele genotype.In some embodiments, the method further comprises, before administering, determining the tumor of the subject for HLA-A allele genotype.In some cases, the subject is determined to have KRAS G12D allele before administering.In some cases, the subject has been genotyped for HLA-A allele before administering.

[0174] The effective amount of pharmaceutical compositions can be described as the amount that is sufficient to achieve predetermined clinical results or beneficial treatment at the required dosage and duration.Effective amount can be delivered in one or multiple administrations.When administration is to a subject that is already known or confirmed to have disease or disease state, the term "therapeutic amount" can be used in relation to treatment, whereas "prophylactically effective amount" can be used to describe administering an effective amount to a subject that is prone to or at risk of developing disease or disease state (e.g., recurrence) as a preventive process.

[0175] Administration can be continuous or intermittent, and parenteral.The composition can be administered locally (for example, intratumorally) or systemically (for example, intravenously).Administration can be for treating a subject who has already been identified as having a recognized condition, disease, or disease state, or for treating a subject who is prone to or at risk of developing such a condition, disease, or disease state.Co-administration with adjunctive therapy can include simultaneous or sequential delivery of multiple agents in any order and in any dosage schedule.

[0176] The methods disclosed herein may further comprise administering one or more additional agents for treating a disease or disorder in combination therapy. For example, in certain embodiments, the combination therapy comprises administering (concurrently, simultaneously, or sequentially) the engineered host cells with an immune checkpoint inhibitor. In some embodiments, the combination therapy comprises administering the host cells with an agonist of a stimulatory immune checkpoint agent. In some embodiments, the combination therapy comprises administering the host cells with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, antibody, or any combination thereof.

[0177] Manufacturing Method In some aspects, the present disclosure provides a method of producing a host cell, the method comprising contacting the host cell with (a) any of the polynucleotides or vectors described herein. In some cases, the method further comprises contacting the host cell with (b) an endonuclease (e.g., a Cas endonuclease, such as a Class II or Type V Cas endonuclease) and (c) a guide RNA compatible with the endonuclease (e.g., compatible with a Class II or Type V Cas endonuclease), wherein the guide RNA is configured to hybridize to an endogenous T cell receptor constant region locus of the host cell before or after the polynucleotide or vector. In some embodiments, the contacting comprises transfection or transduction. In some embodiments, the transfection comprises electroporation. In some embodiments, the T cell receptor constant region locus is the TRAC, TRBC1, or TRBC2 locus. In some embodiments, the guide RNA comprises a sequence having at least 80% sequence identity to any of the guide RNA sequences listed in Table 1. In some embodiments, the guide RNA comprises a pattern of modifications according to any of the guide RNA sequences listed in Table 1. In some embodiments, the Class II, Type V Cas endonuclease is a Type VA Cas endonuclease. In some embodiments, the Class II, Type V Cas endonuclease comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 117. In some embodiments, the host cell comprises an immune cell or a precursor thereof.In some embodiments, the immune cells comprise T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In some embodiments, the immune cells comprise T cells, wherein the T cells comprise CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, or any combination thereof.

[0178] Cells can be engineered to contain or express extracellular binding proteins, additional polypeptides disclosed herein, and / or to reduce expression of endogenous TCR genes. For example, using cell engineering techniques disclosed herein and / or known to those of skill in the art, cells can be modified to contain recombinant nucleic acids encoding the extracellular binding proteins of the present disclosure and / or introduce modifications that reduce expression of endogenous TRAC, TRBC1, and / or TRBC2, thereby generating host cells (such as engineered T cells).

[0179] The method can include contacting a cell with a recombinant nucleic acid, or a vector containing the recombinant nucleic acid, under conditions that allow for uptake of the recombinant nucleic acid by the cell. The recombinant nucleic acid can include a nucleotide sequence that encodes an extracellular binding protein or a component thereof disclosed herein. In some cases, the recombinant nucleic acid is utilized to alter the genome of the cell.

[0180] A recombinant nucleic acid can be a substance, the molecule of which comprises or consists essentially of nucleotides linked in a chain. Non-limiting examples of recombinant nucleic acids include circular nucleic acids, DNA, single-stranded DNA, double-stranded DNA, genomic DNA, plasmids, nanoplasmids, plasmid DNA, viral DNA, minicircles (e.g., lacking a bacterial origin of replication), and RNA.

[0181] A recombinant nucleic acid can include, for example, one or more homology arms containing a sequence complementary to a genomic DNA sequence targeted for insertion (e.g., via homologous recombination). A recombinant nucleic acid can include one or more promoter regions, barcodes, restriction sites, cleavage sites, endonuclease recognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites can be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance characteristics (e.g., antibiotic resistance for selection), or any combination thereof. A recombinant nucleic acid can also include transcriptional or translational regulatory sequences, such as one or more promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A linkers, and / or polyadenylation signals.

[0182] Recombinant nucleic acids can be assembled by various methods, for example, by automated solid-phase synthesis. Recombinant nucleic acids can be constructed using standard solid-phase DNA / RNA synthesis. Recombinant nucleic acids can also be constructed using synthetic procedures. Recombinant nucleic acids can be synthesized manually or in a fully automated manner. In some cases, the synthetic procedure can involve a 5'-hydroxyl oligonucleotide that is first converted to the corresponding 5'-H-phosphonate monoester, which can then be oxidized in the presence of imidazole to an activated 5'-phosphorimidazolidate, and finally reacted with pyrophosphate on a solid support. This procedure can include a post-synthesis purification step, such as PAGE, HPLC, MS, or any combination thereof. Recombinant nucleic acids can be purchased commercially.

[0183] The recombinant nucleic acids described herein can be modified, in some cases, to make them less immunogenic and more stable for transfection into cells.

[0184] For targeted integration, the recombinant nucleic acid sequence to be inserted can be flanked by homology arms comprising sequences that are complementary to the genomic DNA sequence targeted for insertion (e.g., via homologous recombination and / or homology-directed repair (HDR)). A double-strand break can be introduced at the target site in the genome, and the homology arms can facilitate the insertion of the recombinant nucleic acid. In some cases, the recombinant nucleic acid can be excised (e.g., via nuclease) from a vector such as a nanoplasmid and inserted into the genome of a cell.

[0185] The recombinant nucleic acid can be inserted into a safe harbor locus. A safe harbor can include a genomic location where the recombinant nucleic acid can integrate and function without substantially disrupting endogenous activity, e.g., with relatively little impact on local or global gene expression. For example, one or more recombinant nucleic acids can be inserted into any one of HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), CCR5, hROSA26, and / or any combination thereof. The recombinant nucleic acid can be inserted into an intergenic region. The recombinant nucleic acid can be inserted into a non-coding region. The recombinant nucleic acid can be inserted intragenic. In some cases, the recombinant nucleic acid can disrupt the gene into which it is inserted (e.g., reduce or eliminate expression of the disrupted gene). The disrupted gene can be, for example, an endogenous TCR gene (e.g., TRAC, TCRB, TCRBC1, TRBC2, TRG, TRD) or an immune checkpoint gene (e.g., PD-1, CTLA-4). The recombinant nucleic acid can be inserted adjacent to or near a promoter.

[0186] Various enzymes can catalyze the generation of double-strand breaks in genome and / or the insertion of foreign DNA into host genome.Non-limiting examples of gene editing tools and techniques include CRISPR system, CRISPR-associated polypeptide (Cas), TALEN, zinc finger nuclease (ZFN), zinc finger-associated gene regulatory polypeptide, meganuclease, Mega-TAL, transposon-based system, natural master transcription factor, epigenetic modification enzyme, recombinase, flippase, transposase, RNA binding protein (RBP), Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0187] CRISPR system can be used to facilitate the insertion of recombinant nucleic acid encoding extracellular binding protein or its components into cell genome.For example, CRISPR system can introduce double-strand break at target site in genome or random site in genome.

[0188] In some cases, the CRISPR system comprises a CRISPR-associated (Cas) protein or Cas nuclease, including a Type I CRISPR-associated (Cas) polypeptide, a Type II CRISPR-associated (Cas) polypeptide, a Type III CRISPR-associated (Cas) polypeptide, a Type IV CRISPR-associated (Cas) polypeptide, a Type V CRISPR-associated (Cas) polypeptide, or a Type VI CRISPR-associated (Cas) polypeptide, a derivative, variant, or functional fragment thereof.

[0189] In some embodiments, the CRISPR system comprises a Class I system or endonuclease (e.g., a Type I, Type III, or Type IV Cas protein). Class I systems can be of the IA, IB, IC, IU, ID, IE, IF, IV-A, IV-B, III-A, III-D, III-C, or III-B subtype.

[0190] In some embodiments, the CRISPR system comprises a class II system or endonuclease (e.g., type II, type V, or type VI). Class II, type II systems can be of the II-A, II-B, II-C1, or II-C2 subtype. Class II, type V systems can be of the VA, V-B1, V-B2, VC, VD, VE, V-F1, V-F1(V-U3), V-F2, V-F3, VG, VH, VI, VK(V-U5), V-U1, V-U2, or V-U4 subtype. Class II, type IV systems can be of the VI-A, VI-B1, VI-B2, VI-C, or VI-D subtype.

[0191] In some embodiments, the Cas protein used in the methods disclosed herein is a Class II endonuclease. In some embodiments, the Cas protein used in the methods disclosed herein is a Class II, Type V Cas endonuclease. In some embodiments, the Cas protein used in the methods disclosed herein is a Class II, Type VA Cas endonuclease.

[0192] Non-limiting examples of Cas proteins that can be used in a CRISPR system include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm CRISPR enzymes include Cas9, Cas9HiFi ... For example, CRISPR enzymes can direct the cleavage of one or both strands within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence, or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs. The Cas protein can be a high-fidelity Cas protein. Alternatives to Streptococcus pyogenes (S. pyogenes) Cas9 can include RNA-guided endonucleases from the Cpf1 family, which exhibit cleavage activity in mammalian cells.

[0193] In some embodiments, the gene editing system comprises a Cas protein, and the system further comprises a guide RNA (gRNA) complexed with the Cas protein. In some embodiments, the gene editing portion comprises an RBP complexed with the gRNA, which can form a complex with the Cas protein. In some embodiments, the gRNA comprises a targeting segment that exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the target polynucleotide. Multiple gRNAs can be used, for example, to simultaneously or sequentially target TRAC, TRBC1, and / or TRBC2.

[0194] In some cases, a dual nickase approach can be used to introduce double-strand breaks. Cas proteins can be mutated at certain amino acids in either nuclease domain, thereby eliminating the activity of one nuclease domain and generating a nickase Cas protein that can generate single-strand breaks. Nickases can also be used with two separate guide RNAs that target opposite strands to generate DSBs within target sites (often referred to as "double nick" or "dual nickase" CRISPR systems).

[0195] A transposon-based system can be utilized to insert a recombinant nucleic acid encoding the extracellular binding protein or a component thereof of the present disclosure into a genome or to disrupt a TCR-encoding gene. A transposon can comprise a recombinant nucleic acid that can be inserted into a DNA sequence. Class I transposons can be transcribed into an RNA intermediate and then reverse-transcribed and inserted into a DNA sequence. Class II transposons can comprise a DNA sequence that is excised from one DNA sequence and / or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector containing a sequence (e.g., including a transgene) flanked by inverted terminal repeats, and (ii) a source of transposase enzyme. The transposon system (e.g., a class II transposon system) can direct the integration of a recombinant nucleic acid sequence encoding an extracellular binding protein or a component thereof, leaving behind the remainder of the vector. The transposon and transposase can be introduced into a cell. In some cases, a vector encoding a transposase and containing the recombinant nucleic acid is introduced into the cell, where the transposase is expressed and mediates the insertion of the transposon into the genome.

[0196] Examples of transposon-based systems that can be used include, but are not limited to, sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the little brown bat (Myotis lucifugus)); mariner (e.g., derived from the genus Drosophila); frog prince (e.g., derived from the leopard frog (Rana pipiens)); Tol2 (e.g., derived from the fish medaka); and spinON.

[0197] In some embodiments, an extracellular binding protein or other polypeptide can be expressed in a host cell without genomic integration of a recombinant nucleic acid encoding the extracellular binding protein or other polypeptide. For example, the extracellular binding protein or other polypeptide can be expressed from an episomal vector, such as DNA, RNA, circular DNA, circular RNA, minicircle, etc. The extracellular binding protein or other polypeptide can be transiently expressed. For example, the expression of the extracellular binding protein or other polypeptide can decrease as the nucleic acid encoding it is degraded. One method of generating a host cell is through the use of a ribonucleic acid (RNA) system, for example, a system that includes delivering one or more recombinant nucleic acids as RNA. In some cases, the use of RNA can minimize DNA-induced toxicity and immunogenicity, which are sometimes observed with the use of DNA.

[0198] In some cases, one or more recombinant nucleic acids of the present disclosure can be randomly inserted into the genome of a cell.For example, recombinant nucleic acid can encode its own promoter, or can be inserted into a position under the control of endogenous promoter.Alternatively or additionally, recombinant nucleic acid can be inserted into a gene, such as the intron of a gene, the exon of a gene, a promoter, or a non-coding region.

[0199] One or more recombinant nucleic acids and / or gene-editing components can be delivered to cells by any suitable method, for example, using any suitable vector. The vector can be or include a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, a transposon, etc. Any vector system can be used, including, but not limited to, a DNA vector, an RNA vector, a ribonucleoprotein vector, a hybrid DNA-RNA vector, a plasmid vector, a nanoplasmid vector, a minicircle vector, a retroviral vector, a lentiviral vector, an adenoviral vector, a poxvirus vector; a herpesvirus vector, and an adeno-associated viral vector. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes, lipid nanoparticles, or poloxamers. Viral vector delivery systems can include DNA viruses and RNA viruses that have either episomal or integrated genomes after delivery to cells. In some cases, one vector is used. In some cases, two vectors are used. In some cases, three or more vectors are used.

[0200] In some cases, a recombinant nucleic acid and / or gene editing component of the present disclosure can be delivered to a cell without the use of a vector. In some cases, one or more recombinant nucleic acids and / or gene editing components of the present disclosure can be delivered to a cell via a vector, and one or more recombinant nucleic acids and / or gene editing components can be delivered without the use of a vector.

[0201] Cells can be genetically engineered ex vivo to contain recombinant nucleic acids encoding extracellular binding proteins and / or modifications that reduce expression of TRAC, TRB, TRBC1, and / or TRBC2. For example, cells can be collected from a subject in one or more blood draws and / or apheresis procedures, modified ex vivo, optionally selected and / or expanded before and / or after genetic modification, and optionally reintroduced into the subject or a different subject by infusion or injection.

[0202] In some cases, cells are genetically engineered in vivo to include the extracellular binding protein of the present disclosure and / or a modification that reduces the expression of endogenous TRAC, TRB, TRBC1, and / or TRBC2.For example, vectors can be used to deliver gene editing components to cells in a subject without removing the cells from the subject.Vectors can be delivered in vivo by administration to individual subjects, for example, by parenteral administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or topical application.

[0203] Methods for introducing gene editing components into cells include, but are not limited to, electroporation, sonoporation, the use of a gene gun, lipofection, calcium phosphate transfection, the use of dendrimers, microinjection, and the use of viral vectors, including adenoviral, AAV, and retroviral vectors.

[0204] Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the Xenon Electroporation System (ThermoFisher Scientific), or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used to deliver nucleic acids into cells. Electroporation parameters can be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple electrical waveform pulse settings, such as exponential decay, time constant, and square wave. Every cell type has a unique optimal electric field strength (E), which depends on the applied pulse parameters (e.g., voltage, capacitance, and resistance). Application of the optimal electric field strength causes electropermeabilization through the induction of a transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, electroporation pulse width, number of pulses, cell density, and tip type can be adjusted to optimize transfection efficiency and / or cell viability.

[0205] Cells can be selected or enriched for the presence or absence of one or more given factors (e.g., cells can be separated based on the presence or absence of one or more factors). Selection techniques include positive and negative selection techniques, such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). In some cases, cells can be selected before gene editing, for example, to enrich for a population of cells disclosed herein (e.g., immune cells, such as T cells or a T cell subset disclosed herein, e.g., γδ T cells or αβ T cells). Cells can be selected after gene editing, for example, to enrich for a population of cells disclosed herein (e.g., host cells that express an extracellular binding protein or additional polypeptide and / or include a modification that reduces expression of endogenous TRAC, TRB, TRBC1, and / or TRBC2). Host cells can be selected or enriched based on a tag or marker, such as an epitope tag. The tag or marker can be attached to the extracellular binding protein. In some embodiments, the tag or marker is not attached to the extracellular binding protein. The tag or marker can be co-expressed with the extracellular binding protein as disclosed herein. The tag or marker can include a reporter gene, such as a fluorescent protein.

[0206] Cells can be selected, enriched, or expanded based on being positive or negative for a given factor. In some embodiments, cells are selected, enriched, or expanded based on being positive for two or more factors. In some embodiments, cells can be selected, enriched, or expanded based on being positive for one or more factors and negative for one or more factors.

[0207] In some cases, a selectable marker is introduced into the cells, e.g., with or as part of a recombinant nucleic acid encoding the extracellular binding protein, such that cells containing the extracellular binding protein or modification express the selectable marker and can be selected, enriched, or expanded. In some cases, the selectable marker is an antibiotic resistance gene, and cells that do not express the antibiotic resistance gene can be killed by treatment with an antibiotic (e.g., to select for or enrich for cells containing the extracellular binding protein). In some embodiments, the selectable marker is an epitope tag.

[0208] Expression of the extracellular binding proteins, TRAC, TRB, TRBC1, and / or TRBC2 of the present disclosure can be quantified by, for example, qPCR, RNA sequencing, Western blot, or flow cytometry.

[0209] In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of recombinant nucleic acid, after gene editing or delivery of recombinant nucleic acid, before selection, after selection, before expansion, after expansion, or a combination thereof. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of recombinant nucleic acid. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after gene editing or delivery of recombinant nucleic acid. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before expansion. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after expansion.

[0210] Illustrative array Table 1: Exemplary nucleotide and polypeptide component sequences described herein TIFF2026504480000004.tif109158TIFF2026504480000005.tif237158TIFF2026504480000006.tif237158TIFF202 6504480000007.tif242158TIFF2026504480000008.tif242158TIFF2026504480000009.tif242158TIFF20265044800 00010.tif242158TIFF2026504480000011.tif242158TIFF2026504480000012.tif240158TIFF2026504480000013.t if242158TIFF2026504480000014.tif243158TIFF2026504480000015.tif242158TIFF2026504480000016.tif242158 TIFF2026504480000017.tif240158TIFF2026504480000018.tif240158TIFF2026504480000019.tif241158TIFF202 6504480000020.tif241158TIFF2026504480000021.tif242158TIFF2026504480000022.tif242158TIFF20265044800 00023.tif237158TIFF2026504480000024.tif236158TIFF2026504480000025.tif241158TIFF2026504480000026.t if240158TIFF2026504480000027.tif235158TIFF2026504480000028.tif240158TIFF2026504480000029.tif113158 [Example]

[0211] Example 1: CRISPR-mediated disruption of Trac / Trbc in immune cells with or without concomitant modification with KRAS peptide-specific binding proteins (general procedure) CRISPR-mediated disruption of TRAC and TRBC was performed using the following protocol. On day 0, 100 x 10^6 CD4+ or CD8+ T cells were thawed and transactivated (1:100 Transact). Cells were cultured in 6-well G-rex plates (approximately 25 x 10^6 cells / well). Approximately 45-55% of cells died by day 2; therefore, the number of cells was calculated according to the conditions required for knockout (KO) on day 2. T cells were cultured in complete T cell medium containing XVivo™ 15 Serum-free Hematopoietic Cell Medium (Lonza, Basel, Switzerland), 2% Immune Cell Serum Replacement (ICSR), 100 IU / mL IL-2, 5 ng / mL IL-7, and +5 ng / mL IL-15.

[0212] On day 1, T cells were transduced with viruses encoding extracellular binding proteins as described herein (e.g., KRAS G12 mutant-specific TCRs, or any of the TCR sequences listed in Table 1) or left to transactivate until day 2. If transduced with lentivirus (LV), cells were counted and transduced at 5-10 x 10^6 cells per condition.

[0213] On day 2, cells were electroporated with ribonucleoprotein complexes (RNPs) to knock out the TRAC and / or TRBC1 / 2 genes (e.g., using guide RNAs in SEQ ID NOs: 113 or 115). For each electroporation, 250 pmol of guide RNA and sufficient nuclease for a 2.5:1 guide RNA-to-nuclease ratio (MG-29) were combined in a total volume of 7.5 μL for 30 minutes at room temperature to form RNP complexes. While the RNP complexes were forming, cells were counted, and 5–10 × 10 cells were used per electroporation for a 100 μL electroporation volume using Neon (ThermoFisher). If cells were transduced on day 1, they were generally not counted again on day 2.

[0214] To set up the Neon electroporator, CTS Xenon electroporation buffer (Thermo, catalog number A4997901) and E2 buffer were first thawed to RT in a BSE. E2 electroporation buffer is a hyperosmolarity buffer commercially available from ThermoFisher Scientific. The required volume of electroporation buffer was then transferred to a microcentrifuge tube or 15 mL tube. Next, 7 mL of complete T cell medium (X-VIVO15 (Lonza, (Lexington, MA) 04-418Q) + 2% PLTGold (Mill Creek Life Sciences (Rochester, MN) PLTGOLD100 GMP)) was added to each well of a 24-well plate used for non-adherent cell growth, commercially available as a Grex plate, and transferred to 37 °C (for culturing the cells after electroporation).

[0215] 5–10 × 10 cells per electroporation condition were then spun down in a 15 mL tube. The medium was carefully aspirated, and the cell pellet was resuspended in 100 μL of electroporation buffer (for single knockout (sKO)) or 95 μL of GE electroporation buffer (for double knockout (dKO)). The total volume recommended for electroporation is 120 μL. For dKO, TRAC RNP was combined with TRBC RNP (7.5 μL each for the required number of conditions). For sKO, 7.5 μL of RNP (after 30 min of incubation) or for dKO, 15 μL of RNP was added to the cells in GE buffer. The cells and RNP were mixed by gently flicking the tube.

[0216] Approximately 100 μL of the mix containing RNP and electroporation buffer was carefully pipetted using a Neon 100 μL pipettor and tip. Care was taken to avoid introducing air bubbles into the pipette tip. If bubbles formed after two or three attempts, the tip was replaced or 10–15 μL of electroporation buffer was added to the tube and the process was repeated. The cell medium was aspirated, and the cell pellet was allowed to stand in the BSE for 15–20 minutes until ready for electroporation.

[0217] 3 mL of E2 buffer was then transferred to a Neon electroporation tube and placed in a tube holder.

[0218] The electroporation pipette with the tip containing the cells and RNP was transferred to a tube containing E2 buffer in a tube holder, and the electroporation protocol on the Neon was performed using the appropriate settings for the electroporator. Exemplary settings for the Neon electroporator are 2300 V, 4 pulses, and a 3 ms pulse width.

[0219] After electroporation, the cells were then transferred to the wells of a 24-well plate (standard tissue culture (TC) plate, which can be adherent or non-adherent) - electroporation ("EP plate") and incubated in BSE at room temperature for approximately 10 minutes. Approximately 1 mL of T cell medium from the G-rex plate was used to wash the cells from the 24-well EP plate and transfer them to the wells in the G-rex plate. The plate was returned to 37°C with 5% CO2.

[0220] Next, peptide dose-dependent responses were examined for cells engineered with the following constructs: pGE106 (encoding TCR2, CD34-IL7R, and CD8α / β), pGE116 (encoding TCR2, CD34-IL7R, and CD8α / β and designed to use the endogenous TRAC promoter), pGE107 (same as pGE106 but with shorter homology arms), and pGE129 (encoding TCR4, CD34-IL7R, and CD8αβ). Responses were assessed by analyzing the percentage of 2A-positive cells expressing CD137 after stimulation with KRAS-G12D peptide. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism® (Figure 22C). The experiment was repeated using cells modified with pGE106, pGE114 (encoding TCR2, CD34, IL7R, CD8α / β, and the SV40 enhancer), and pGE116, and further modified to have a single knockout at the TRAC locus or a double knockout at the TRAC and TRBC loci (Figure 22D). Similar results were observed for cells cocultured with antigen-presenting cells (APCs) instead of the cognate peptide (Figure 22E), and when cocultured with certain cancer cell lines (Figures 2F-2M).

[0221] T cells modified with constructs encoding either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, via either electroporation or LVV-mediated transduction, exhibited in vitro proliferation when cocultured with various KRAS-G12D-positive cell lines (Figures 22N-22T).

[0222] Cytotoxic activity was assessed for cells engineered to express TCR2, CD34IL7R, and CD8α / β, or TCR2, CD58IL7R, and CD8α / β, and plots of red fluorescence measured over time were generated by live cell imaging. The results demonstrate that the cells are more effective in controlling HuCCT1 tumor cells than controls (Figure 22U).

[0223] Example 2: Editing efficiency and specificity of CRISPR-mediated disruption of Trac / Trbc in immune cells CD4+ and CD8+ T cells were subjected to CRISPR-mediated destruction of TRAC and TRBC as described in Example 1.

[0224] CD4 / CD8 T cells electroporated with gRNAs targeting the TRAC and TRBC genes were evaluated for CD3 expression on the cell surface and for indels at the on-target genomic loci. High editing efficiency was achieved at both the TRAC and TRBC loci using a CRISPR-associated nuclease type V called MG29-1. Editing activity was comparable to that of the CRISPR / Cas9 system (Figures 19A and 19B).

[0225] Five hundred and ninety computationally predicted potential off-target sites (OTs) with up to six mismatches with the target sequence were selected across the TRAC and TRBC gRNAs. Indel activity was assessed at each of these potential OTs in primary T cells treated with TRAC and TRBC gRNAs. High on-target editing efficiency was observed, but no off-target activity was detected above background, exceeding the assay's quantitation limit (0.05%) (Figure 20).

[0226] Oligo-capture analysis was performed in primary T cells to further evaluate the specificity of these nuclease / gRNA combinations. A small number of potential off-target sites (OTs) were identified, each of which had at least 100-fold fewer barcodes than the target site and multiple mismatches (>9) to the target site, suggesting that these OTs were unlikely to be true positives (Figure 21).

[0227] The frequency of potential translocation outcomes between the TRAC and TRBC loci was assessed in edited primary T cells using digital PCR (dPCR) and karyotyping. Editing T cells with TRAC / TRBC gRNA resulted in a low frequency of translocation events. No enrichment of translocation events was observed over time in culture (Figures 22A and 22B).

[0228] Next, peptide dose-dependent responses were examined in cells modified with the following constructs: pGE106 (encoding TCR2, CD34-IL7R, and CD8α / β), pGE116 (encoding TCR2, CD34-IL7R, and CD8α / β and designed to use the endogenous TRAC promoter), pGE107 (same as pGE106 but with shorter homology arms), and pGE129 (encoding TCR4, CD34-IL7R, and CD8αβ). Responses were assessed by analyzing the percentage of 2A-positive cells expressing CD137 after stimulation with KRAS-G12D peptide. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism® (Figure 22C). The experiment was repeated using cells modified with pGE106, pGE114 (encoding TCR2, CD34, IL7R, CD8α / β, and the SV40 enhancer), and pGE116, and further modified to have a single knockout at the TRAC locus or a double knockout at the TRAC and TRBC loci (Figure 22D). Similar results were observed when cells were cocultured with antigen-presenting cells (APCs) instead of the cognate peptide (Figure 22E) and when cocultured with certain cancer cell lines (Figures 22F-22M).

[0229] T cells modified with constructs encoding either TCR2 and CD8α / β, or TCR2, CD34-IL7R, and CD8α / β, via either electroporation or LVV-mediated transduction, exhibited in vitro proliferation when cocultured with various KRAS-G12D-positive cell lines (Figures 22N-22T).

[0230] Cytotoxic activity was assessed for cells engineered to express TCR2, CD34IL7R, and CD8α / β, or TCR2, CD58IL7R, and CD8α / β, and plots of red fluorescence measured over time were generated by live cell imaging. The results demonstrate that the cells are more effective in controlling HuCCT1 tumor cells than controls (Figure 22U).

[0231] Example 3: Activation of T cells expressing candidate KRAS-G12D-binding TCRs This example demonstrates the activation of T cells expressing the TCRs disclosed herein by target cells pulsed with KRAS-G12D peptide.

[0232] Paired TCRα / β sequences from identified clonotypes were assembled and synthesized as P2A-linked expression cassettes, as described, for example, in Hilgarth and Lanigan, MethodsX 7 (2020) 100759, and lentivirally transduced into reporter Jurkat cells (Nur77-GFP-Jurkat) expressing GFP under the control of the Nur77 locus to indicate TCR activation. Peptide dose-dependent responses for each TCR were assessed by analyzing GFP expression after overnight culture with A11 target cells pulsed with decreasing concentrations of peptide. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism®. TCR091 exhibited the highest affinity in two replicate assays (Figures 1A and 1B).

[0233] Example 4: Target cell killing by T cells expressing candidate KRAS-G12D-specific TCRs This example demonstrates killing of target cells displaying KRAS-G12D by T cells expressing the TCRs disclosed herein, with or without knockout of TRAC and TRBC.

[0234] Red fluorescent Hpaf-II cells, a KRAS-G12D-expressing tumor cell line transduced to express HLA-A11, were transduced with TCR32-transduced CD8+ cells harboring wild-type TRAC and TRBC loci. + T cells, or TCR32-transduced TRAC / TRBC double knockout (dKO) CD8 +CD8 T cells were co-cultured at a 3:1 effector:target cell ratio. Total red object integrated intensity (a measure of tumor cell volume) was assessed over time. Red fluorescence was measured by live cell imaging using an IncuCyte S3 microscope and software package, a live cell analysis system. + T cell cytotoxicity is indicated by a reduction in total red target cell area per well compared to untreated wells. To assess TCR-mediated tumor cell lysis by transduced T cells in the persistent presence of antigen, additional tumor cells were added at 72 and 140 hours. Tumor cell reduction was observed when co-cultured with TCR-transduced cells (Figure 2).

[0235] In similar experiments, primary CD4+ and CD8+ T cells transduced with an additional KRAS G12D-specific T cell receptor, with or without TRAC and TRBC knockout, were evaluated for cytotoxicity against Hpaf-II cells. In this case, a single rechallenge with tumor cells at 72 hours was included. Improved T cell killing activity was generally observed in TRAC / TRBC dKO T cells (Figure 4B) compared with cells with intact wild-type TRAC / TRBC (Figure 4A).

[0236] The impact of TRAC and TRBC knockout on cancer cell killing was also assessed using HuCCT1 (cholangiocarcinoma; Figures 4C-4F), Hpaf-II (pancreatic adenocarcinoma; Figures 4G-4I), and Panc1 (pancreatic ductal adenocarcinoma; Figure 4J) target cells, which display KRAS G12D, an additional KRAS-G12D-specific T cell receptor. In some cases, due to tumor cells reaching confluence, cell death was observed at later time points in tumor cell-only or mock-treated conditions. KRAS-G12D-specific engineered T cells with knockout of the endogenous TRAC / TRBC gene demonstrated enhanced killing of multiple cell lines, as summarized in Figure 4K.

[0237] Example 5: Activation of T cells expressing a candidate KRAS-G12D TCR is enhanced by knockout of TRAC and TRBC This example demonstrates that knockout of TRAC and TRBC can enhance activation of T cells expressing the candidate KRAS G12D-specific TCRs disclosed herein.

[0238] Primary CD4+ and CD8+ T cells were transduced with KRAS-G12D-specific T cell receptors (TCR69, TCR79, TCR80, TCR81, or TCR91) and edited to knock out TRAC and TRBC, or the TRAC and TRBC genes were left unmodified and expanded for several days. Engineered T cells were incubated with decreasing concentrations of G12D peptide, and T cell responses were assessed by measuring CD137 expression. The data demonstrate that knockout of the TRAC / TRBC locus improved TCR avidity in cells sourced from two donors (Figures 3A and 3B).

[0239] In additional experiments, the effects of TRAC and TRBC knockout were evaluated on primary CD4+ and CD8+ T cells transduced with additional KRAS-G12D-specific TCRs. Double knockout improved TCR avidity for multiple TCRs, as shown in Figure 3C (corresponding to the EC50 values ​​in Table 2 and the Cmax values ​​in Table 3), Figure 3D (corresponding to the EC50 values ​​in Table 4), and Figure 3E (corresponding to the EC50 values ​​in Table 5). TCR2, TCR4, TCR5, and TCR6 exhibited the lowest EC50 values ​​in this screen. In many cases, substantially lower EC50 values ​​(e.g., 3- to 5-fold improvement) were observed with TRAC and TRBC knockout.

[0240] Table 2. T cell activation EC50 (nM) values ​​of T cells expressing candidate KRAS G12D-specific TCRs with and without knockout of TRAC and TRBC. TIFF2026504480000030.tif26128

[0241] Table 3. Cmax (%) values ​​of T cells expressing candidate KRAS G12D-specific TCRs (without knockout of TRAC and TRBC) TIFF2026504480000031.tif26128

[0242] Table 4. T cell activation EC50 (nM) values ​​of T cells expressing candidate KRAS G12D-specific TCRs with and without knockout of TRAC and TRBC. TIFF2026504480000032.tif41128

[0243] Table 5. T cell activation EC50 values ​​in nM of T cells expressing candidate KRAS G12D-specific TCRs with and without knockout of TRAC and TRBC. TIFF2026504480000033.tif41128

[0244] Example 6: TCR selectivity To assess potential TCR off-target activity, CD4+ / CD8+ T cells expressing the TCRs disclosed herein were cultured overnight with a panel of 191 positional scanning peptides containing all possible amino acid substitutions at each position of the cognate KRAS G12D peptide (Figure 5A). Secreted IFNγ levels were used to identify TCR cross-reactivity to promiscuous positions. Potentially antigenic peptides were then matched to the human proteome using ScanProsite (prosite.expasy.org / scanprosite / ) to predict potential in vivo off-targets. Peptides that elicited responses greater than 10% of the maximum signal were considered positive in this assay. As summarized in Table 6, few or no potential off-targets were identified for TCR91 (Figure 5B), TCR2 (Figure 5C), TCR4 (Figure 5D), and TCR5 (Figure 5E), which recognize G12D.

[0245] (Table 6) TIFF2026504480000034.tif31128

[0246] In vitro assays were performed to assess the specificity of TCR091 for the KRAS-G12D peptide and potential off-target reactivity with RASL11B, an unrelated off-target interaction partner of the KRAS-G12D TCR. TCR91-transduced primary CD4+ / CD8+ T cells carrying wild-type TRAC / TRBC loci or TRAC / TRBC dKO were cultured overnight with decreasing concentrations of KRAS-G12D or RASL11B peptides, and CD137 expression was assessed by flow cytometry. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism®. T cell activation in response to the KRAS-G12D peptide was greater in dKO T cells than in TRAC / TRBC wild-type cells (Figure 6). Additionally, only a very low level of response (approximately 10%) was detected with RASL11B.

[0247] Example 7: Activation and target cell killing by T cells expressing a candidate KRAS-G12D TCR is enhanced by knockout of TRAC and TRBC This example demonstrates that knockout of TRAC and TRBC can enhance activation and thereby target cell killing of T cells expressing the candidate KRAS G12D-specific TCRs disclosed herein.

[0248] TCR91-transduced primary CD4 cells harboring wild-type TRAC / TRBC locus, TRAC single knockout (sKO), or TRAC / TRBC dKO + / CD8 +T cells were expanded for 10 days and treated with decreasing concentrations of G12D peptide. T cell activation was measured using CD137 expression as assessed by flow cytometry. Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using GraphPad Prism®. TCR91-transduced TRAC / TRBC dKO cells showed enhanced activation compared to WT and TRAC single KO cells (Figure 7).

[0249] In further experiments, TCR91-transduced TRAC / TRBC dKO cells showed increased activation compared to WT and TRBC sKO cells (Fig. 11).

[0250] To assess the impact of single or double TRAC / TRBC knockouts on target cell killing, TCR91-transduced primary CD4+ / CD8+ T cells harboring wild-type TRAC / TRBC loci, TRAC single knockout (sKO), or TRAC / TRBC dKO were expanded for 10 days and cocultured with HpafII cells at a 3:1 effector:target cell ratio. Cytotoxic activity was assessed by generating plots of red fluorescence measured over time by live-cell imaging. Results demonstrate that TRAC / TRBC dKO cells are more effective at controlling HpafII tumor cells than either TRAC sKO or TRAC / TRBC wild-type cells (Figure 8). Enhanced cytotoxicity was also observed in assays using Panc-1 tumor cells as target cells (Figure 9).

[0251] In further experiments, the cytotoxicity of dKO was compared with that of a single knockout (sKO) of TRBC. TCR91-transduced primary CD4+ / CD8+ T cells harboring wild-type TRAC / TRBC locus, TRBC sKO, or TRAC / TRBC dKO were expanded for 10 days and cocultured with fluorescent HpafII cells at a 3:1 effector:target cell ratio. TRAC / TRBC dKO TCR91-transduced cells were more effective in controlling HpafII tumor cells than either TRBC sKO or TRAC / TRBC wild-type cells (Figure 12).

[0252] In a separate assay, primary CD4+ and CD8+ T cells were transduced with a KRAS-G12D-specific T cell receptor (TCR-91) at a transduction efficiency of 35% (Figure 13A) or 70% (Figure 13B) with or without TRAC and TRBC knockout. After several days of expansion, the engineered cells were cocultured with HpafII cells at a 3:1 effector:target cell ratio. Results demonstrated that TRAC / TRBC dKO cells were more effective at controlling HpafII tumor cells than TRAC / TRBC wild-type cells.

[0253] Similar studies were performed using Panc-1 tumor cells as target cells. Primary CD4+ and CD8+ T cells were transduced with KRAS-G12D-specific TCR91 with or without TRAC and TRBC knockout at a transduction efficiency of 35% (Figure 14A) or 70% (Figure 14B). After several days of expansion, the engineered cells were cocultured with fluorescent Panc-1 cells at a 10:1 effector:target cell ratio. Cytotoxic activity was assessed using an IncuCyte assay. The results demonstrate that TRAC / TRBC dKO cells are more effective in controlling Panc-1 tumor cells than TRAC / TRBC wild-type cells.

[0254] Example 8: Tetramer analysis of KRAS-G12D TCR with knockout of TRAC and / or TRBC This example demonstrates enhanced surface expression and peptide-MHC binding of the KRAS-G12D TCR disclosed herein in knockouts of TRAC and TRBC.

[0255] TCR91-transduced primary CD4+ / CD8+ T cells harboring wild-type TRAC / TRBC loci, TRBC single knockout (sKO), or TRAC / TRBC dKO were expanded for 10 days. Cell surface expression of the correctly paired G12D TCR and its functionality were assessed using fluorochrome-labeled tetramers of MHC-peptide complexes. Increases in the percentage of cells staining positive for peptide-MHC binding (Figure 10A) and mean fluorescence intensity (MFI, reflecting the average signal per cell; Figure 10B) were observed in cells with knockout of the endogenous TCR-encoding gene, with a greater increase in TRAC / TRBC dKO.

[0256] In a similar study, a comparison was made to TRAC single knockout. TCR91-transduced primary CD4+ / CD8+ T cells harboring a wild-type TRAC / TRBC locus, a TRAC single knockout (sKO), or a TRAC / TRBC dKO were expanded for 10 days and assessed for cell surface expression of the correctly paired G12D TCR via binding of fluorochrome-labeled MHC-peptide tetramers. Increased percentages of cells staining positive for peptide-MHC binding (Figure 15A) and mean fluorescence intensity (MFI, reflecting the average signal per cell; Figure 15B) were observed in cells with endogenous TRAC / TRBC knockout (dKO) compared with WT or TRAC sKO cells.

[0257] First generation CD4 + / CD8 +Additional experiments with T cells also demonstrated enhanced surface expression and peptide-MHC binding of three KRAS G12D-specific TCRs (Table 7; see also representative flow plots in Figure 15C, where the percentage of tetramer-positive, 2A-positive cells was 48.6% in the dKO versus 13.5% without knockout).

[0258] (Table 7) TIFF2026504480000035.tif31128

[0259] Example 9: In vivo efficacy of T cells expressing the candidate KRAS-G12D TCR with knockout of TRAC and TRBC This example demonstrates the in vivo efficacy of T cells expressing the KRAS G12D-specific TCR disclosed herein with knockout of TRAC and TRBC.

[0260] Nod scid gamma (NSG) mice were subcutaneously implanted with CL40 colon adenocarcinoma cells, randomized (n = 5 per group), and treated 9 days after implantation with 10 x 10^6 T cells (control T cells with knockout of endogenous TRAC and TRBC, or T cells expressing the KRAS G12D-specific TCR disclosed herein with co-expression of the CD8 co-receptor (CD8αβ) and knockout of endogenous TRAC and TRBC) administered intravenously. A 1:1 ratio of CD4+ and CD8+ T cells was administered. The engineered cells demonstrated robust antitumor efficacy, as demonstrated by tumor volume control (Figure 16A). During the study period, 100% complete responses and 100% survival were observed for the group receiving T cells expressing the KRAS G12D-specific TCR with dKO, whereas none of the animals receiving control T cells survived (Figure 16B).

[0261] Example 10: KRAS G12D TCR T cells with enhanced IL7R signaling exhibit enhanced proliferation and tumor cell killing This example demonstrates enhanced proliferation and tumor cell killing via the engineered cells disclosed herein.

[0262] CD4+ T cells and CD8+ T cells were engineered to contain (i) a KRAS G12D-specific TCR disclosed herein; (ii) a CD8 co-receptor (CD8αβ); (iii) knockout of endogenous TRAC and TRBC expression; and / or (iv) a chimeric fusion protein comprising the IL-7 receptor intracellular signaling domain, transmembrane domain, and extracellular domain (e.g., the extracellular domain from CD34, CD58, or CD80).

[0263] The engineered cells were co-incubated with HuCCT1 tumor cells and assessed for STAT5 phosphorylation, proliferation, and tumor cell killing. Cells with the chimeric IL7R fusion protein exhibited increased STAT5 phosphorylation (Figure 17A; indicating IL7R activity), proliferation (Figure 17B), and tumor cell killing (including upon multiple rechallenge; Figure 17C) compared to cells lacking the chimeric IL7R fusion protein.

[0264] 1x10^7 engineered cells were administered intravenously to mice 9 days after subcutaneous inoculation of HuCCT1 tumor cells. Inclusion of a chimeric IL-7R fusion protein further improved the antitumor response, resulting in a complete response (Figure 17D).

[0265] Example 11: Non-viral transgene integration can achieve high efficiency of transgenesis and improve functionality of engineered T cells This example provides a comparison of lentiviral versus non-viral targeted transgene integration and the functionality of the resulting engineered T cells.

[0266] For non-viral knock-in and simultaneous knock-out of TRAC and TRBC, the CRISPR system was used with electroporation of Cas-gRNA RNP to generate double-strand breaks in the TRAC and TRBC loci. Nanoplasmid was used to provide a template for insertion of an expression cassette into the TRAC locus via homology-directed repair (HDR). RNP and nanoplasmid DNA were electroporated 48 hours after T cell activation. Electroporation was performed using Xenon. Non-viral knock-in resulted in integration efficiencies of up to 44% in primary CD4+ and CD8+ T cells (Figure 18A).

[0267] Following non-viral knock-in or lentiviral transduction, killing of Panc-1 tumor cells was assessed using the IncuCyte assay described for Figure 2, incubating cells at an effector-to-target ratio of 10:1. Enhanced killing was observed for engineered T cells generated by non-viral knock-in (Figure 18B). Enhanced killing was also observed with the introduction of a chimeric fusion protein containing the IL-7 receptor intracellular signaling domain as described in Example 10.

[0268] In additional experiments, killing of HuCCT1 tumor cells was assessed using an IncuCyte assay following non-viral knock-in or lentiviral transduction. T cells engineered via non-viral knock-in demonstrated improved tumor cell killing, including after rechallenge with fresh tumor cells 48 hours later (Figure 18C).

[0269] Example 12: Illustrative Non-Viral Transgene Integration Protocol This example provides an illustrative method for non-viral knock-in of a transgene / expression cassette in combination with knockout of TRAC, TRBC1, and / or TRBC2.

[0270] Materials: (i) T cell donor of interest; (ii) Nanoplasmid (5 mg / mL stock); (iii) X-Vivo-15, cytokines; (iv) 6-well and 24-well GREX plates; (v) CTS Xenon Electroporation Buffer (Gibco, Catalog No. A4997901) or CTS Xenon Genome Editing Buffer (Gibco, Catalog No. A4998001); (vi) 96-well flat-bottom plates (assay plates); (vii) 96-well U-bottom plates (staining plates); (viii) Miltenyi T-cell activating Transact (Miltenyi, Catalog No. 130-111-160); (ix) antibodies against CD4, CD8, TCRab, P2A, Live / Dead, tetrameric peptide conjugates; (x) BD CytoFix; and (xi) 10x BD Perm / Wash.

[0271] Day 0: Thawing of cells Fresh medium for thawing T cells contains either X-vivo 15 + 2% Immune Cell Serum Replacement (ICSR) + IL2 (100 units), IL7 (5 ng / mL), and IL15 (5 ng / mL); or X-vivo 15 + IL2 (100 units), IL7 (500 units), and IL15 (500 units).

[0272] The cells are subjected to rapid thawing and then resuspended in complete medium (e.g., 10 mL final volume). The T cells are counted (Celleca, AOPI staining) to determine the number and viability of the cells. The T cells are spun at 300 x g for 5 minutes, then the medium is removed and the cell pellet is replaced with 1 mL of fresh medium.

[0273] Transfer the resuspended cells to wells on a 6-well GREX plate (approximately 24 mL of medium per well). Activate the T cells by supplementing the wells with 1:100 Miltenyi Transfectant (e.g., 250 µL per 25 mL of medium) and incubate overnight.

[0274] Day 2: Knock-in electroporation RNPs are complexed at room temperature for 20-30 minutes using a 2:1 nuclease-to-guide molar ratio: (a) sgRNA (300 μM stock): 2.1 μL; (b) MG-21 nuclease (6.93 mg / mL stock): 5.4 μL; (c) total RNPs per reaction: approximately 7.5 μL. While the RNPs are complexing, donor T cells from the bottom of the GREX plate are resuspended, pooled, and counted. T cells are collected and spun down at 300 x g for 5 minutes. Neon electroporation reactions use 5 x 10^6 cells per reaction, and Xenon electroporation reactions use 50 x 10^6 cells per reaction. Post-EP medium is X-vivo medium + IL 2, 7, and 15, containing 5% ICSR. If performing a double knockout, mix each RNP 1:1 (15 μL per EP reaction for Neon and 150 μL for Xenon) for easier pipetting. Because the buffer can be toxic to cells, pellet the cells and resuspend them in EP buffer only when ready to electroporate.

[0275] Neon electroporation option: Reaction conditions include: (a) 100 μL electroporation buffer to resuspend cells; (b) 7.5 μL per RNP; (c) KI nanoplasmid (20 μg) if used at the concentration of interest: 4 μL plasmid; and (d) electroporation: 2300 V, 4 pulses, 3 ms pulse width.

[0276] Place the electroporation tube into the electroporation unit, fill the tube with 3-5 mL of E2 buffer, and connect the conductor. Attach the electroporation tip to the syringe (press down firmly; the gold-plated conductor rod should not bend more than 30% further than the unpressed syringe). Use the syringe to gently pipette the sample and draw it into the tip. There should be no air bubbles at the top of the tip, as this will interfere with electroporation. Once loaded into the Neon Tip, transfer it to the casing and fit the syringe snugly onto the tube. Activate the Neon electroporator; it will beep once to confirm selection and again when complete. Press the tip into a 24-well flat-bottom plate and allow the cells to rest for 10 minutes. After 10 minutes, remove 1 mL of medium from the final destination well and resuspend the rested cells. Incubate the cells at 37°C for growth, and count the cells on day 4.

[0277] Xenon electroporation option: Reaction conditions include: (a) 800 µL electroporation buffer to resuspend cells; (b) 200 µL master editing mix (see below); and (c) EP protocol: 2300 V, 4 pulses, 3 ms pulse width, 500 ms pulse interval.

[0278] Make a 200 μL master mix of RNP, buffer (same as for Neon electroporation), and nanoplasmid (or other nucleic acid molecule): 75 μL per RNP, 40 μL nanoplasmid (200 μg, if used), and qs to 200 μL with electroporation buffer. Pellet the cells at 300 x g for 5 minutes. Resuspend the pellet in 800 μL of buffer. Add 200 μL of editing master mix to the 800 μL. Transfer the entire volume (1 mL) into a Xenon One Shot cartridge by removing the top and filling the unit. The cell solution should be slightly convex to ensure contact with the conductor at the top of the cartridge. Insert the Xenon One Shot cartridge into the instrument and turn on the electroporator. The Xenon electroporator will begin the pulse and indicate when the process is complete. Upon removal, the cells are allowed to rest in the cartridge for 10 minutes before being transferred into 40 mL of Xvivo15+5% ICSR+cytokines and incubated at 37° C. The next day, 60 mL of fresh medium is added to the wells.

[0279] Example 13: KRAS G12D-specific TCRs expressing CD8ab co-receptors and chimeric cytokine receptors have enhanced activity in vitro and in vivo T cells engineered with T cell receptors (TCRs) that recognize epitopes derived from intracellular oncogenic drivers, such as mutant KRAS, the most frequently altered gene in human cancers, are likely to induce durable responses in patients with solid tumors. T cells were engineered by non-viral targeted knock-in (KI) of the TCR alpha constant chain (TRAC) locus to express a multicistronic cassette containing 1) a high-affinity TCR specific for the KRAS G12D mutation, 2) a CD8αβ co-receptor, and 3) a chimeric cytokine receptor. The engineered T cells exhibited cytotoxicity against endogenously expressing HLA-A*11:01+ / KRAS G12D+ cell lines in vitro and mediated robust antitumor activity in vivo. The engineered cells also demonstrated a favorable safety profile for KRAS G12D-specific TCRs and gene editing reagents. This data supports the planned clinical development of these engineered T cells as a novel non-viral knock-in (KI) TCR-engineered T cell therapy for KRAS mutant solid tumors.

[0280] Autologous CD4+ and CD8+ T cells were engineered using the Type V CRISPR-Cas system (Goltsman et al., Novel Type VA CRISPR effectors are active nucleases with expanded targeting capabilities, CRISPR J., 2020; Lamothe et al., Novel CRISPR-associated gene-editing systems discovered in metagenomic samples enable efficient & specific genome engineering. CRISPR J. 2023) to knock out the endogenous TRAC locus and simultaneously knock in (KI) a non-virally delivered transgene within the TRAC locus (Schober et al., Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function, Nature Biomed. Engg., 2019). These cells (G12D TCR-T cells) were engineered to express i) a high-avidity HLA-A*11:01-restricted TCR specific for the KRAS G12D mutant peptide, ii) a CD8α / β co-receptor that drives coordinated CD8 / CD4 T cell responses by enabling CD4 stimulation that promotes functional survival of CD8+ T cells, and iii) an interleukin receptor, ILR, a fusion protein that promotes antitumor activity through increased T cell proliferation and survival.

[0281] The engineered cells were tested for functional avidity through binding to the KRAS G12D peptide (Figure 23, left). As demonstrated by T cell activation using CD137 as an activation marker, the engineered cells specifically bound to the KRAS G12D peptide even at subnanomolar concentrations. The engineered cells were then tested against HuCCT1 (bile duct tumor) cells (Figure 23, right). The engineered cells exhibited robust cytotoxicity even after rechallenge with tumor cells 48 and 120 hours after administration.

[0282] The engineered cells were then administered intravenously (IV) to NOD SCID gamma (NSG) mice initially inoculated subcutaneously with either HuCCT1 tumor cells (Figure 24, left) or CL40 colon cancer tumor cells (Figure 24, right). The engineered cells demonstrated robust tumor cell control in vivo in response to both tumor cell lines.

[0283] The engineered cells were also incubated with a library of X-scan peptides. Cell activation was measured to screen for KRAS G12D TCR recognition motifs (Figure 25). The recognition motifs were then referenced to human peptides to identify any potential cross-reactivity. The engineered cells showed undetectable levels of cross-reactivity with peptides even at a concentration of 500 nM, demonstrating that the engineered cells are highly specific for KRAS G12D and have a low risk of cross-reactivity to other human peptides in vivo.

[0284] The gene editing reagents used to engineer the cells were further tested for any potential off-target activity. First, potential off-target sites were identified in silico (Figure 26A) and using oligo-capture analysis in primary T cells (Figure 26B). For each potential off-target, the presence of insertions and / or deletions in the engineered cells was assessed using targeted sequencing arrays. The results showed that the engineered cells exhibited high on-target activity and had insignificant amounts of off-target activity. This demonstrated that the gene editing reagents were highly specific in producing engineered cells.

[0285] The efficiency of the non-viral knock-in (KI) process was also tested (Figure 27). Results showed that over 40% of T cells showed integration of the transgene. This was also tested using small (research) scale and scaled-up (10-fold) size batches, each of which showed robust expansion growth kinetics.

[0286] T cells from the patient donor were then engineered using the KI process used above. The subsequent engineered patient cells were then tested for KI efficiency, growth kinetics, and functionality (FIG. 28). The results showed that the engineered patient cells functioned very similarly to healthy donor T cells.

[0287] Example 14: Non-viral targeted knock-in of KRAS G12D-specific TCR, CD8αβ, and chimeric cytokine receptors at the TRAC locus outperforms lentiviral-based engineering of T cells T cells engineered with T cell receptors (TCRs) offer a high potential for stably expressing and targeting oncogenic driver mutations, inducing durable responses in patients with solid tumors. Viral vectors, including lentiviruses (LVVs), have been the standard modality for transgene delivery for T cell therapy, but are extremely limited in their manufacturing time, cost, and cargo size. In contrast, non-viral targeted gene knock-in (KI) overcomes these limitations and substantially reduces manufacturing complexity. Here, we compared primary human T cells engineered using either the LVV or KI process. Primary human T cells were engineered using either process to express a TCR that recognizes the KRAS G12D mutant peptide presented on HLA*A11:01, a CD8α / β co-receptor, and a chimeric interleukin receptor (ILR). We developed and optimized a nonviral manufacturing process using a novel CRISPR-Cas12a system to knock in a transgene cassette into the TRAC locus and simultaneously knock out endogenous TCR1. This nonviral KI platform edited cells with high efficiency, and KI-engineered TCR T cells performed better in functional assays than LVV-engineered cells. Together, these data support the utility of a nonviral genetic KI approach and its planned incorporation into clinical development.

[0288] Engineered T cells for the KI process were engineered using the Metagenomi-derived MG29-1 nuclease (Goltsman, DSA et al. Novel Type VA CRISPR Effectors Are Active Nucleases with Expanded Targeting Capabilities, CRISPR J., 2020) of the Type V CRISPR-Cas system to knock out endogenous TCRs and drive transgene integration via homologous recombination.

[0289] The transgene construct used for the LVV process contained a murine stem cell virus (MSCV) promoter driving the expression of transcripts containing a TCR that recognizes the KRAS G12D mutant peptide presented on HLA*A11:01, a CD8α / β co-receptor, and a chimeric interleukin receptor (ILR) (Figure 29). First, the effect of the size of the transgene construct on transduction efficiency and viral titer was tested for the LVV process. The results (Figure 29) showed that viral titer decreased with increasing transgene size (left), and transduction efficiency decreased with increasing transgene size (right). This demonstrated that the efficiency of the LVV process was limited by the transgene size.

[0290] The transgene construct used for the KI process contained an elongation factor-1 alpha (EF-1α) promoter driving the expression of transcripts containing a TCR that recognizes the KRAS G12D mutant peptide presented on HLA*A11:01, a CD8α / β co-receptor, and a chimeric interleukin receptor (ILR) (Figure 30A). The knock-in process was designed to insert the transgene construct into the endogenous TRAC gene via CRISPR / Cas-driven homologous recombination repair. The efficiency of the KI process was then tested (Figure 30B). The results showed that this optimized KI process reached an efficiency of over 50% while producing a T cell population similar to that of the LVV process.

[0291] Next, T cells engineered using the KI process were tested for binding to the KRAS G12D tetramer (Figure 31). The results demonstrated that the KI process exhibited improved binding to the KRAS G12D tetramer compared to the LVV process. This indicates that the EF-1α promoter was able to drive higher TCR expression despite fewer expected copies of the transgene construct per cell.

[0292] Next, T cells engineered using the KI process were tested for functional avidity (Figure 32). KI-processed T cells again performed superiorly compared to LVV-processed T cells, with higher functional avidity resulting in higher T cell activation, as measured by the T cell activation marker CD137.

[0293] The T cells engineered using the KI process were then tested in vivo in NSG mice against T cells engineered using the LVV process. Ten days after subcutaneous inoculation of NSG mice with HuCCT1 tumor cells, 5 million CD4 / CD8 engineered T cells derived from either the KI process or the LVV process were administered intravenously to the mice (Figure 33). The results demonstrated that the T cells engineered using the KI process also showed superior tumor control compared to T cells engineered using the LVV process.

[0294] A promoterless transgene construct for the KI process was also developed alongside the original construct (Figure 34). This allows the endogenous TRAC promoter to be used to drive expression of the transgene construct transcript instead of the EF-1α promoter. This promoterless transgene construct was then tested against the EF-1α transgene construct (Figures 35-37). The results demonstrated that use of the endogenous TRAC promoter resulted in less transgene expression (Figure 35), lower functional avidity (Figure 36), and less in vivo activity (Figure 37) compared to the EF-1α promoter.

[0295] Example 15: Phase I Study of Autologous CD8+ and CD4+ Engineered T Cell Receptor T Cells in Subjects with Advanced or Metastatic Solid Tumors A Phase 1, first-in-human (FIH), multicenter, open-label study of KRAS G12D TCR T cells will be conducted. The study will consist of a dose-escalation and dose-expansion portion. The study will enroll adult male and female subjects with KRAS G12D-positive advanced or metastatic cancer who are HLA-A*11:01-positive and have progressed on or are intolerant to at least one line of prior systemic therapy for their current malignancy.

[0296] Approximately 10 study sites for dose escalation and approximately 40 sites for dose expansion are planned, enrolling approximately 100 subjects.

[0297] Subjects will be followed in the main study for up to 24 months or until disease progression is documented. After post-treatment discontinuation, subjects will be followed for up to 15 years after the last KRAS G12D TCR T cell infusion. The study will consist of three periods: pre-treatment, treatment, and post-treatment.

[0298] The pre-treatment period consisted of the following: KRAS G12D-HLA Early Screening: Subjects may consent to enter into early screening to determine their KRAS G12D and HLA status. This screening can occur any time prior to eligibility screening. Eligibility Screening: Starting with signing the Master Consent Form, up to 30 days to determine full eligibility for enrollment. Leukapheresis will proceed only upon confirmation of eligibility. Leukapheresis: Leukapheresis is performed in eligible subjects, followed by a manufacturing period of approximately 28 days. Bridging therapy may be administered during the manufacturing period. Baseline assessment: Assessment before lymphocyte-depleting chemotherapy (LDC).

[0299] The treatment period consisted of the following: Lymphocyte-depleting chemotherapy with cyclophosphamide 500 mg / m^2 from days -6 to -3 and fludarabine 30 mg / m^2 from days -6 to -3. Alternative regimens are permitted. KRAS G12D TCR T cell infusion on day 1. o Subjects may be re-treated if they meet the re-treatment criteria.

[0300] The post-treatment period consisted of the following: Post-Treatment Follow-Up: To monitor safety and anti-tumor activity for up to 24 months after KRAS G12D TCR T cell infusion or until disease progression (PD), whichever occurs first. After completion of post-treatment follow-up, at post-treatment follow-up discontinuation, and at the end-of-study visit, subjects will consent to a 15-year long-term follow-up study.

[0301] The study will continue until all subjects discontinue study participation (including complete withdrawal of consent), die, are lost to follow-up, or are transferred to another long-term follow-up protocol if one becomes available. A schematic of the study is shown in Figure 38.

[0302] Study duration The study is expected to take approximately 18 months to enroll the dose-escalation portion and approximately 12 months to enroll the dose-expansion portion. Subjects will undergo post-treatment follow-up (PTFU) for up to 24 months or until documented disease progression. After completing the PTFU period or documented PD, subjects will transition to LTFU for up to 15 years from their last KRAS G12D TCR T-cell infusion. Study objectives and endpoints are shown in Table 8.

[0303] Table 8: Study objectives and evaluation items TIFF2026504480000036.tif153170TIFF2026504480000037.tif231170TIFF2026504480000038.tif175170

[0304] KRAS G12D TCR T cell construct: An autologous KRAS G12D TCR T cell product targeting the KRAS G12D mutation is used herein. The KRAS G12D TCR T cell product used is an autologous CD4+ and CD8+ TCR T cell therapy that expresses a multicistronic cassette consisting of 1) a high-avidity TCR specific for the KRAS G12D mutation, 2) a constitutively active IL-7Rα fused to the CD34 ectodomain, and 3) a CD8α / β co-receptor. T cells are engineered using the CRISPR-Cas12a system to knock-in (KI) a transgene into the TRAC locus and simultaneously knock-out (KO) the endogenous TRAC locus. Non-viral delivery to T cells is performed using a nanoplasmid encoding five transgenes (KRAS G12D TCR α and β chains, constitutively active IL-7Rα, and CD8 α and β chains).

[0305] Overall study design: This is a Phase 1, first-in-human (FIH), multicenter, open-label study of KRAS G12D TCR T cells, consisting of dose-escalation and dose-expansion parts.

[0306] The trial will enroll adult male and female subjects with KRAS-positive advanced or metastatic cancer, who are HLA-A*11:01 positive, and who have progressed on or are intolerant to at least one line of prior systemic therapy for their current malignancy. Approximately 10 study sites for dose escalation and approximately 40 sites for dose expansion are planned, enrolling approximately 100 subjects.

[0307] Determining sample size The total number of subjects planned to be enrolled in both arms of the study is approximately 100 and includes: Dose escalation: up to 20 subjects Dose expansion: up to 4 cohorts, with a maximum of 20 subjects per cohort, for a total of approximately 80 subjects

[0308] The sample size for dose escalation will be selected to be consistent with that of a typical Phase 1 trial and will be calibrated based on simulations to obtain reasonable operating characteristics. The sample size for dose expansion will be selected based on a Bayesian optimal Phase 2 (BOP2) design (Zhou et al., 2017, Stat Med. 36(21):3302-14), as described below, with a pre-specified Type I error and power.

[0309] Dose titration / titration part A total sample size of up to 20 subjects will be enrolled in the dose-finding / escalation portion of the study. Each dose cohort will consist of approximately 2-4 subjects, with a time lag of at least 28 days between the KRAS G12D TCR T cell infusion of the first and second subject in each new dose level (DL) cohort.

[0310] All subjects in a cohort must complete the full dose-limiting toxicity (DLT) period before the next cohort can enter the treatment period.

[0311] The optimal biological dose (OBD) will be found using the BOIN12 trial design (Lin et al., 2020, JCO Precis Oncol. 2020;4), which uses utility to quantify dose desirability in terms of toxicity-efficacy tradeoffs and adaptively assigns subjects to the dose with the highest estimated desirability.

[0312] Dose desirability (i.e., risk-benefit tradeoff) is quantified using utilities. The utilities assigned to each possible efficacy-toxicity outcome are listed in Table 9, where higher values ​​indicate more desirable outcomes (0 and 100 indicate the least desirable and most desirable outcomes, respectively). Let u1, . . . , u4 represent these utilities. Let p1, . . . , p4 represent the corresponding probability of observing each of the possible toxicity-efficacy outcomes, given dose j. Then, the average utility for dose j is: u j =p1u1+p2u2+p3u3+p4u4

[0313] Higher values ​​of u j indicates the higher desirability of dose j in terms of risk-benefit trade-off.

[0314] Table 9. Utility table for dual efficacy and toxicity endpoints TIFF2026504480000039.tif25160

[0315] To protect subjects from toxic and / or ineffective doses, BOIN12 uses two dose acceptability criteria to determine which doses can be used to treat a subject. A dose is considered acceptable and eligible for treating a subject if it meets the following safety and efficacy criteria: Safety: Pr(πT>0.3|data) <0.95 Efficacy: Pr(πE<0.25|data) <0.9 where πT and πE are the true DLT rate and efficacy rate, respectively. These two conditions ensure that an acceptable dose cannot be excessively toxic or ineffective. Unacceptable doses are excluded from the study. If a dose is excluded due to toxicity, all doses higher than that dose are also excluded. The goal is to identify the OBD, defined as the dose that is both acceptable and has the highest desirability. A maximum of 20 subjects will be enrolled, with cohort sizes of 2 to 4 subjects.

[0316] After dose-finding is complete, the OBD will be selected as the dose that is tolerable and has the highest estimated efficacy based on the isotonic estimation method described in Lin et al. (2020). The recommended phase II dose (RP2D) will be selected based on design recommendations and the overall benefit-risk evidence.

[0317] Dose expansion part After the RP2D is determined, additional subjects will be enrolled in dose expansion cohorts to further evaluate the safety and efficacy of KRAS G12D TCR T cells. The dose expansion portion may consist of up to four cohorts, with a maximum of approximately 20 subjects per cohort. Subjects participating in the dose expansion portion will be treated at the RP2D determined at the end of the dose escalation portion. Cohorts in the dose expansion portion will be indication-specific and may include advanced or metastatic non-small cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and any other tumor type harboring the KRAS G12D mutation that warrants the potential antitumor activity of KRAS G12D TCR T cells. Both toxicity and futility will be monitored using the BOP2 design (Zhou et al., 2017).

[0318] Futility monitoring will be conducted separately for each of the four indication-specific cohorts. The sponsor will monitor the 6-month overall response rate (ORR) using a BOP2 design when approximately 10 subjects are treated. In addition, for subject safety, toxicity will be monitored for every 10 subjects in the combined cohorts, up to a maximum of 80 subjects.

[0319] Analysis population Data are presented and analyzed in the following populations: Screening: All subjects who sign informed consent for eligibility screening Enrollment: All subjects undergoing leukapheresis KRAS G12D TCR T cell therapy: All enrolled subjects will receive KRAS G12D TCR T cells in a targeted DL Intent-to-Treat (ITT): All enrolled subjects Efficacy evaluable: All subjects in the KRAS G12D TCR T-cell treatment population with at least one post-baseline disease response assessment, or discontinuation due to PD, or death before the first post-treatment disease response assessment Safety analysis: All subjects who received any low-dose chemotherapy (LDC) and / or KRAS G12D TCR T cells The ITT population will be used for summary demographic and baseline characteristics, biomarker and efficacy analyses, and subgroup analyses will be completed, including efficacy and safety analyses of subgroups based on receipt of bridging chemotherapy. The primary safety analysis will be based on the safety analysis population. b. Details of the analysis will be prospectively specified in a Statistical Analysis Plan (SAP).

[0320] statistical analysis General Considerations Summary statistics (number of non-missing values, mean, median, standard deviation, minimum, and maximum for continuous variables, and number and percentages for categorical variables) will be provided, including demographic and baseline values, and safety measures, where appropriate. No formal statistical inference will be performed on safety parameters. No imputation will be performed for missing data.

[0321] The safety summary displays results for each part separately and for all subjects combined.

[0322] Primary endpoint safety The primary safety endpoint will be assessed via the occurrence of adverse events (AEs), serious adverse events (SAEs), and DLTs. AEs will be coded using the most recent version of the Medical Dictionary for Regulatory Activities (MedDRA). AE data will be summarized using descriptive statistics.

[0323] Secondary endpoints: Secondary objectives are preliminary anti-tumor response, as measured by ORR (partial response (PR) + complete response (CR)) according to RECIST v1.1, duration of response (DOR), progression-free survival (PFS), time to response (TTR), clinical benefit rate (CBR), and overall survival (OS). ORR and its 95% confidence interval (CI) will be estimated.

[0324] Exploratory endpoints: Details of the analysis of exploratory endpoints will be described in the SAP. Depending on sample availability and the ability to derive parameters, PK parameters of KRAS G12D TCR T cells may be calculated, including maximum concentration, time to maximum concentration, AUC, and half-life. Exposure-safety and exposure-efficacy analyses may be considered.

[0325] Given the small size of the study, the correlative biomarker study was underpowered for formal statistical analysis and is therefore not expected to yield direct conclusions but instead inform hypotheses for future research. Where applicable, exploratory analyses will be described using standard statistical methods, taking into account the sample size at each stage of the study.

[0326] DLT definition and stopping rules: Monitoring for dose-limiting toxicity During the dose escalation part of the study, subjects will be monitored for DLTs, which are defined as AEs assessed as probably or related to KRAS G12D TCR T cells that occur within the first 28 days after the first KRAS G12D TCR T cell infusion and that meet the following criteria: 1. Any grade 5 death unrelated to the underlying disease and at least possibly related to KRAS G12D TCR T cells 2. Grade 4 or 5 CRS occurring during any treatment 3. Any treatment-emergent grade 3 CRS that does not resolve to grade 2 within 7 days 4. Grade 3 or higher neurotoxicity that does not resolve to Grade 2 within 72 hours 5. Grade 3 or higher immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) 6. Grade 3 or higher allergic reaction associated with KRAS G12D TCR T cell infusion 7. Any treatment-emergent autoimmune toxicity, grade 3 or greater 8. Grade 3 or greater organ toxicity (cardiac, skin, gastrointestinal, hepatic, pulmonary, renal / genitourinary) occurring within 30 days of cell infusion and not due to pre-existing or underlying malignancy Any Grade 3 or greater KRAS G12D TCR T-cell related non-hematologic toxicity that does not resolve to Grade 2 or less within 7 days 9. Grade 3 thrombocytopenia with bleeding 10. Grade 3 or higher hematologic toxicity that does not resolve to Grade 2 or lower within 7 days and is not attributable to LDC administration 11. Any other clinically significant toxicity related to KRAS G12D TCR T cells that is deemed by the investigator to represent a DLT and does not meet the criteria above The following AEs are considered DLTs: do not have . 12. Grade 3 Fatigue 13. Grade 3 endocrinopathy (thyroid, pituitary, and / or adrenal insufficiency) managed with or without systemic corticosteroids and / or hormone replacement therapy with resolution of symptoms 14. Grade 3 hypertension that can be controlled with medical treatment 15. Transient, asymptomatic, and clinically insignificant Grade 3 laboratory abnormalities 16. Vitiligo or alopecia of any AE grade

[0327] DLT grading will be performed according to NCI-CTCAE version 5.0. For ICANS and CRS, the American Society for Transplantation and Cellular Therapy Consensus Grading will be used (Lee et al., 2019). When applicable, the DLT evaluation period will be extended to track ongoing TEAEs until resolution of the event or confirmation that the event is a DLT.

[0328] Stopping rules: The Safety Monitoring Committee (SMC) will monitor cumulative adverse events (AEs) in all subjects and will hold the study in the event of recurrent, clinically significant, or late treatment-related toxicity. The SMC will evaluate whether the study should be discontinued based on excessive toxicity as outlined in the BOIN12 clinical trial design under dose tolerability criteria. If no dose level meets safety tolerance, the study will be terminated. Indication-specific stopping rules for futility will apply during dose expansion.

[0329] Test stopping criteria: The SMC will evaluate whether the study should be discontinued based on excessive toxicity. If no dose level meets safety acceptability criteria, the study will be terminated. The SMC will meet periodically as needed.

[0330] The trial may be paused or stopped if any subject experiences any of the following SAEs after KRAS G12D TCR T cell infusion: Death occurring within 30 days of study drug administration, excluding deaths due to disease progression Death within 30 days of infusion that is unrelated to the underlying disease and is at least possibly related to KRAS G12D TCR T cells Two grade 4 or higher SAEs occurred in two study subjects Over 33% of study subjects experienced Grade 3 events regardless of study phase Any grade 4 hypersensitivity reaction / anaphylaxis; or infection associated with a positive sterility test Life-threatening (grade 4) toxicity caused by KRAS G12D TCR T cells that is unexpected, unmanageable (i.e., does not resolve to grade 3 or less within 10 days), and unrelated to bridging therapy between leukapheresis and LDC

[0331] If paused, the study will be discontinued until an appropriate assessment of the cause of toxicity has been determined and a corrective plan established, if necessary. In addition, a study may be terminated for the following reasons: Any subject develops uncontrolled proliferation of KRAS G12D TCR T cells that leads to malignant tumors Observation of unexpected significant risks to the subject Failure to register subjects at an acceptable rate plans to modify, suspend, or discontinue development of KRAS G12D TCR T cells; The sponsor, IRB / IEC, or SMC determines that continuing the trial may jeopardize the safety of the subjects.

[0332] Inclusion / Exclusion Criteria KRAS-HLA screening inclusion criteria Subjects must meet all of the following inclusion criteria to be eligible to participate in KRAS-HLA screening. 1. Be at least 18 years old 2. Understand and are willing to provide written informed consent 3. Able and willing to comply with the requirements of the clinical trial protocol 4. Histologically confirmed, advanced or metastatic unresectable solid tumors 5. Previously determined KRAS G12D mutation status and HLA-A*11:01 allele, or willingness to provide a sample for analysis of KRAS and HLA-A*11:01

[0333] Study eligibility screening inclusion criteria All of the following inclusion criteria must be met prior to leukapheresis. 1. Be at least 18 years old 2. Understand and are willing to provide written informed consent 3. Able and willing to comply with the requirements of the clinical trial protocol, including the availability of a dedicated caregiver 4. Histologically confirmed, advanced or metastatic unresectable solid tumors 5. Positive for KRAS G12D mutation 6. The subject has the HLA-A*11:01 allele 7. Have progressed on or are intolerant to at least one line of prior standard systemic therapy for their current malignancy. Subjects with tumors with known actionable molecular alterations must have progressed on directed molecular therapy. For CRC: Subjects with genomic abnormalities, including but not limited to BRAFV600E mutations and HER2 amplification, for which FDA-approved targeted therapies are available, must have received prior treatment with an applicable FDA-approved targeted therapy. Subjects whose tumors are deficient mismatch repair (dMMR) / microsatellite instability-high (MSI-H) must have received an immune checkpoint inhibitor prior to enrolling in this study. For NSCLC: Subjects with genomic abnormalities for which an FDA-approved targeted therapy is available must have received prior treatment with an applicable FDA-approved targeted therapy. Subjects for whom such treatment is appropriate must have received treatment with an FDA-approved checkpoint inhibitor with or without chemotherapy consistent with the FDA-approved label. Any other solid tumor, including PDAC: Subjects with genomic abnormalities for which an FDA-approved targeted therapy is available must have received prior treatment with an applicable FDA-approved targeted therapy. Subjects whose tumors are dMMR / MSI-H must have received an immune checkpoint inhibitor prior to enrolling in this study. 8. Measurable disease per RECIST v1.1. Note: Previously irradiated or locoregionally treated lesions may be considered target lesions if they progress after treatment. 9. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 10. Adequate organ and bone marrow function based on the following laboratory values: Absolute neutrophil count (ANC) ≥ 1000 / mm3 without granulocyte colony-stimulating factor support (filgrastim within 7 days or peg-filgrastim within 14 days of the screening test used for subject eligibility) Absolute lymphocyte count (ALC) ≥ 200 / mm 3 Platelets ≥ 75,000 / mm3 without transfusion within the previous 7 days 3 Hemoglobin ≥ 8.0 g / dL (≥ 80 g / L); transfusion is permitted without a transfusion within the previous 7 days Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3 × upper limit of normal (ULN) If liver metastasis is present, ALT and AST ≤ 5 × ULN Total bilirubin ≤ 1.5 x ULN, or ≤ 3 x ULN in the presence of documented Gilbert syndrome Albumin ≥ 2.5 g / dL (≥ 25 g / L) Prothrombin time / international normalized ratio (INR) or partial thromboplastin time (PTT) test <1.5 × ULN; abnormal values ​​are permitted if the subject is taking low-dose low-molecular-weight heparin Creatinine clearance ≥ 50 mL / min as determined by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) or 24-hour urine clearance NOTE: Eligible laboratory test results, including test results from standard of care tests, may be used for study enrollment if within 7 days. Eligible tests may be used to proceed to leukapheresis if the test was performed within 7 days of leukapheresis. Able and willing to comply with contraceptive guidelines and local regulations regarding contraceptive use. Agree to abstain from breastfeeding while participating in the study. Refrain from tissue donation, including egg / sperm donation or any other tissue / blood / organ donation, for at least one year after the last KRAS G12D TCR T cell infusion.

[0334] LDC inclusion criteria All of the following criteria must be met at the study baseline visit within 72 hours prior to the LDC. ECOG performance status of 0 or 1 Adequate organ and bone marrow function within 72 hours prior to the planned LDC, based on the following criteria: ANC≧1000 / mm 3 ALT and AST ≤ 3 × ULN ■ If liver metastasis is present, ALT and AST ≤ 5 × ULN Total bilirubin ≤ 1.5 x ULN, or ≤ 3 x ULN in the presence of documented Gilbert syndrome Creatinine clearance ≥ 50 mL / min by CKD-EPI or 24-hour urinary clearance Subjects of childbearing potential must have a negative serum or urine pregnancy test within 72 hours prior to LDC Bridging toxicities, if applicable, must have resolved to Grade 1 or baseline prior to LDC If subjects receive immunotherapy during bridging therapy, they should have evidence of prior progression on a PD-1 or PD-L1 checkpoint inhibitor; progression occurring within the first 8 weeks of treatment with these agents should be confirmed by a second CT scan at least 4 weeks apart.

[0335] KRAS G12D TCR T cells Eligibility for initial and retreatment infusions Prior to KRAS G12D TCR T cell infusion, subjects will undergo clinical evaluation and determination of suitability to proceed with administration of AFNT. All of the following inclusion criteria must be met prior to KRAS G12D TCR T cell infusion. ECOG performance status of 0 or 1 Adequate organ and bone marrow function based on the following criteria: ALT, AST ≤ 3 × ULN ■ If liver metastasis is present, ALT and AST ≤ 5 × ULN Total bilirubin ≤ 1.5 x ULN, or ≤ 3 x ULN in the presence of documented Gilbert syndrome Creatinine clearance ≥ 50 mL / min by CKD-EPI or 24-hour urinary clearance Note: To be eligible for retreatment, subjects must have tolerated the initial infusion without a DLT.

[0336] Eligibility Exclusion Criteria Study eligibility exclusion criteria Any subject meeting any of the following criteria will be excluded from study participation: Any systemic cytotoxic chemotherapy, investigational drug, or any antineoplastic agent (including small molecules and I / O compounds) from a previous treatment regimen or clinical trial within 5 half-lives or 14 days of screening, whichever is shorter Any prior gene therapy using an integrating vector Previous allogeneic stem cell transplant or prior organ transplant History of treatment for a primary immunodeficiency, autoimmune, or inflammatory disease, including inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, or Graves' disease The following autoimmune conditions are permitted: vitiligo, alopecia, hypothyroidism on stable hormone replacement therapy, stable adrenal insufficiency with or without low-dose prednisone, psoriasis / eczema not requiring systemic treatment, or any other condition deemed not clinically significant. Use of prohibited steroids or immunosuppressants within 14 days prior to screening Primary brain tumors Untreated central nervous system (CNS) metastatic disease, leptomeningeal disease, or spinal cord compression. Subjects previously treated for CNS metastases who are radiographically and neurologically stable and weaned from steroids for at least 4 weeks prior to enrollment are eligible. Surgery or catheter-based intervention, e.g., transarterial chemoembolization or percutaneous coronary intervention, within 4 weeks prior to screening · Uncontrolled significant concurrent or recent illness, including but not limited to the following conditions: Subjects with clinically significant pulmonary dysfunction, as determined by medical history and physical examination, should undergo pulmonary function testing. Subjects with a forced expiratory volume in 1 second (FEV1) less than 55% or a diffusing capacity for carbon monoxide (DLCO) less than 40% will be excluded. Significant cardiovascular abnormalities, as defined by any one of the following: uncontrolled congestive heart failure or hypertension, clinically significant hypotension, symptomatic coronary artery disease, or a documented ejection fraction (EF) of less than 45% as assessed by echocardiogram or multigated acquisition scan (MUGA). Any subject with an EF between 45% and 49% must be cleared by a cardiologist to be eligible for the study. Uncontrolled active bacterial, viral, fungal, or mycobacterial infections that do not respond to antibiotics, antimycotics, or antifungal agents, and long-term oral treatment with any of these agents Pregnant or breastfeeding subjects Previously identified allergy, hypersensitivity, or known contraindication to cyclophosphamide, fludarabine, or any other agent related to LDC or AFNT-212 Diagnosis of another malignancy within 2 years prior to screening The following diagnoses are permitted: superficial non-melanoma skin cancer, T1-T2a, clinically localized low-risk prostate cancer defined as a tumor with histologic grade group 1 (Gleason score ≤ 6) on biopsy and serum PSA < 10 ng / ml, cervical intraepithelial carcinoma, and resected lobular or ductal carcinoma in situ that is considered cured and has not been treated with systemic therapy; subjects with other resected early-stage cancers with a low risk of recurrence may be eligible after documented discussion with the medical monitor. Seropositive for hepatitis B surface antigen (HBsAg) and / or hepatitis B core antibody (HBcAb) Seropositive for Hepatitis C antibodies. Subjects with a positive Hepatitis C antibody test may be eligible, after discussion with the medical monitor, if Hepatitis C virus (HCV) RNA is undetectable by quantitative HCV RNA assay. Known human immunodeficiency virus (HIV) infection Psychiatric or other comorbid conditions that affect compliance with study procedures, as determined by the investigator

[0337] Lymphocyte-depleting chemotherapy exclusion criteria Any subject who meets any of the following criteria will be excluded from the LDC. If applicable, the LDC may be postponed and the investigator should contact the medical monitor: LDC with broad-field radiation therapy or chemoradiotherapy within 4 weeks or palliative limited-field radiation therapy within 2 weeks Systemic antitumor therapy prior to LDC Any cytotoxic chemotherapy, investigational agent, or any antineoplastic agent (including small molecules and I / O agents) from a previous treatment regimen or clinical trial within 14 days Nitrosoureas or mitomycin C within 6 weeks of LDC Use of steroids or immunosuppressants within the last 14 days before LDC The following treatments are permitted: intranasal, inhaled, topical, or localized steroid applications; systemic corticosteroids at doses equivalent to 10 mg / day or less of prednisone; and steroids as premedication for contrast allergies. Uncontrolled significant concurrent or recent illness or active infection that has not responded to antibiotic, antifungal, or antiviral treatment Live attenuated vaccines within 30 days prior to LDC

[0338] Leukapheresis, and LDC and post-infusion procedures Leukapheresis Following the eligibility screening assessment, a leukapheresis collection will be performed for eligible subjects. If technical issues arise during the procedure or processing of the leukapheresis product, the subject may undergo a second collection. The subject must continue to meet the eligibility requirements for a repeat leukapheresis.

[0339] Subjects who are ineligible for venovenous apheresis may choose to have a percutaneous central venous access catheter inserted to assist in this collection.

[0340] If necessary, anti-cancer bridging therapy is permitted for disease control after apheresis and while KRAS G12D TCR T cells are manufactured.

[0341] Requirements after LDC and KRAS G12D TCR T cell infusion Hospitalization of study subjects following LDC or KRAS G12D TCR T cell infusion is not required under this protocol. The decision to refer study subjects to inpatient services for post-infusion observation not related to the management of acute adverse events (AEs) is at the investigator's discretion and will be made in accordance with institutional guidelines and processes.

[0342] Study subjects should remain within a two-hour transportation distance to the study center for 28 days following KRAS G12D TCR T cell infusion to allow for consultation at the study center in the event of toxicity. Subjects may alternatively utilize accommodations in accordance with facility practices.

[0343] Dedicated caregiver All subjects must have a dedicated caregiver for 28 days following KRAS G12D TCR T cell infusion, who will assist subjects with ICE assessments and temperature checks at home.

[0344] Lifestyle Considerations Subjects will be given a Study Wallet Card with emergency contact information for the investigator and / or study staff and instructed to carry it with them at all times.

[0345] Test treatment Table 10 Lymphocyte-depleting chemotherapy TIFF2026504480000040.tif67160IMP=Investigational Drug; LDC=Lymphodepleting Chemotherapy; IV=Intravenous; NIMP=Non-Investigational Drug

[0346] Upon notification from the sponsor that KRAS G12D TCR T cells are available, LDC should be scheduled to begin 6 days before the scheduled infusion date. KRAS G12D TCR T cells must be on-site before LDC begins. Details of LDC treatment are provided in Table 10. Details of KRAS G12D TCR T cell infusion are provided in Table 11. Both LDC and KRAS G12D TCR T cell infusion will be administered at the study site by qualified personnel in either an outpatient or inpatient setting, according to site procedures. If the investigator believes it is in the subject's best interest to begin LDC before the KRAS G12D TCR T cells arrive at the site and has been notified that KRAS G12D TCR T cells are available in a timely manner, the investigator should contact the sponsor to discuss the rationale and obtain approval.

[0347] If the subject received bridging therapy or was imaged more than 28 days prior to the planned Day 1 KRAS G12D TCR T cell infusion, the baseline imaging assessment must be repeated (after completion of bridging therapy, if applicable) before the start of the LDC.

[0348] Subjects will undergo LDC from day -6 to day -3 (4 days). However, if the subject is deemed by the investigator to be sufficiently lymphodepleted (absolute lymphocyte count 0-15 uL) on day 3 of LDC, day 4 may be omitted.

[0349] (Table 11) KRAS G12D TCR T cell injection TIFF2026504480000041.tif156160IMP=Investigational Drug

[0350] Subjects must complete the LDC prior to KRAS G12D TCR T cell infusion. Enrolled subjects will not begin study treatment until the KRAS G12D TCR T cell product has been manufactured, tested, cleared for release, and received at the clinical site.

[0351] If the assigned target dose range cannot be met, the subject may, at the investigator's discretion and after a documented benefit-risk discussion with the medical monitor, receive the manufactured KRAS G12D TCR T-cell IMP. Safety and toxicity data will be collected. However, the subject will no longer be evaluable for DLT, will not be counted in the assigned dose cohort, and will need to be replaced.

[0352] At the investigator's discretion, KRAS G12D TCR T cells may be given more than two days after completion of LDC. However, if the infusion delay exceeds seven days after LDC, this should be discussed with the medical monitor. If there is a significant delay (e.g., more than two months) in KRAS G12D TCR T cell infusion, LDC may be repeated.

[0353] KRAS G12D TCR T cell infusion should be postponed if the subject exhibits any of the following on the scheduled infusion day: Eligibility for KRAS G12D TCR T cell infusion not met Suspected or active systemic infection (subjects with suspected / active infection must have at least 24-hour negative cultures with appropriate antibiotics or a negative rapid viral panel) Hypotension requiring vasopressor support The need for supplemental oxygen to maintain saturation above 91%

[0354] Retreatment criteria Subjects who received KRAS G12D TCR T cells and experienced a partial response may receive a second infusion of KRAS G12D TCR T cells at the investigator's discretion. Subjects who achieved a transient CR and subsequently progressed within the PTFU period may also be considered for retreatment. Retreatment must be supported by a benefit-risk assessment and justified by efficacy and toxicity data, as well as identified biological activity from the previous dose. Subjects may be retreated after review of the patient's condition and discussion with the medical monitor.

[0355] To be eligible for retreatment, subjects must meet all of the criteria defined below. At least 8 weeks after the first injection Confirmed disease progression (expansion cohort only) ECOG performance status of 0 or 1 - No anti-cancer drugs have been administered since the AFNT-212 infusion Adequate organ and bone marrow function ANC≧1000 / mm 3 ALT, AST ≤ 3 × ULN ■ If liver metastasis is present, ALT and AST ≤ 5 × ULN Total bilirubin ≤ 1.5 x ULN, or ≤ 3 x ULN in the presence of documented Gilbert syndrome Creatinine clearance ≥ 50 mL / min by CKD-EPI or 24-hour urinary clearance Subjects of childbearing potential must have a negative serum or urine pregnancy test within 72 days prior to the LDC

[0356] In addition, subjects must have tolerated the initial infusion of KRAS G12D TCR T cells without experiencing any DLTs. Retreatment must be administered at the same dose level as the initial infusion of KRAS G12D TCR T cells. If the availability of cell product for retreatment is limited, a lower dose may be considered after discussion with the medical monitor. If retreatment occurs within 2 months of the initial infusion of KRAS G12D TCR T cells, LDCs will not be administered. If retreatment occurs more than 2 months after the initial infusion of KRAS G12D TCR T cells, the decision to administer LDCs is at the investigator's discretion; however, subjects must meet the LDC eligibility criteria. The same LDC regimen used before the initial KRAS G12D TCR T cell infusion should be used before retreatment.

[0357] AEs following re-treatment with KRAS G12D TCR T cells will be collected and reported but will not be used in the DLT analysis.

[0358] Timing of KRAS G12D TCR T cell infusion for dose escalation cohorts During the dose escalation part of the study, up to four dose levels of KRAS G12D TCR T cells may be evaluated.

[0359] For each dose escalation cohort, KRAS G12D TCR T cells will be administered with a 28-day staggered time between dosing of the first and second subject at each new dose level to allow for event monitoring.

[0360] Anti-cancer bridging therapy Experimental treatments may not be used in bridging therapy. If bridging therapy is used, it must be completed at least 14 days prior to LDC. Subjects may not receive bridging therapy for re-treatment with KRAS G12D TCR T cells.

[0361] Concomitant medications and procedures required Anti-infective prophylaxis may be initiated at the start of the LDC or after the LDC at the investigator's discretion and in accordance with the recommendations of respective professional associations and local institutional guidelines.

[0362] In some cases, tocilizumab, or another anti-interleukin (IL)-6 therapy, may be required to treat toxicities such as CRS. Anti-IL-6 therapy must be available on-site prior to subject infusion.

[0363] See currently approved prescribing information. Additionally, glucocorticoids such as dexamethasone or methylprednisolone may be utilized for ICANS and CRS.

[0364] Cytopenias may be managed with growth colony-stimulating factors, packed red blood cells (PRBCs), and platelet transfusions according to institutional guidelines and / or PI discretion. Leukofilters are recommended for PRBC transfusions.

[0365] Fever in the presence of neutropenia should be managed according to local institutional guidelines.

[0366] The schedule of study activities and procedures is shown in Table 12. The schedule of biomarker and pharmacokinetic activities is shown in Table 13.

[0367] Table 12. Schedule of study activities and procedures TIFF2026504480000042.tif248137TIFF2026504480000043.tif248146TIFF2026504480000044.tif248139Abbreviations: AE = adverse event; CRS = cytokine release syndrome; CT = computed tomography; D = day; DLT = dose-limiting toxicity; ECG = electrocardiogram; ECHO = echocardiogram; ECOG = Eastern Cooperative Oncology Group; EoT = end of treatment; G6PD = glucose-6-phosphate dehydrogenase; HLA-A *11:01 = human leukocyte antigen-A; HRQoL = health-related quality of life; ICANS = immune effector cell-associated neurotoxicity syndrome; ICE = immune effector cell-associated encephalopathy; ICF = informed consent form; KRAS = Kirsten rat sarcoma viral oncogene homolog; LDC = lymphocyte-depleting chemotherapy; LTFU = long-term follow-up; M = month; MRI = magnetic resonance imaging; MUGA = multigated acquisition scan; O2 = oxygen; PD = progressive disease; PET = position emission tomography; PK = pharmacokinetics; PTFD = post-treatment follow-up discontinuation; RECIST = Response Evaluation Criteria in Solid Tumors; SOCBP = childbearing potential; TLS = tumor lysis syndrome; yrs = years All assessments and sample collections will be performed prior to LDC and AFNT-212 infusion unless otherwise stated. Additional assessments to evaluate subject safety and efficacy may be performed at unscheduled visits and will be recorded on the eCRF. For further details regarding sample collection and shipping to the central laboratory, refer to the Study Laboratory Manual. a. The ICF must be signed before any protocol-specific assessments are performed unless otherwise specified. Separate ICFs for the STFU and LTFU periods will be signed at the M24 / PTFD visit. b. The baseline assessment must be performed within 72 hours (3 days) of the LDC. The baseline procedure may also be performed on the day of the LDC. c. For KRAS and HLA testing, samples should be set aside for potential diagnostic development. d. The registration evaluation must be performed within 30 days of leukapheresis unless otherwise specified. Radiological imaging within 45 days of leukapheresis is acceptable. e. Subjects will record their daily oral temperature at home twice daily (approximately 8 hours apart) for 14 days after AFNT-212 infusion and once daily for 28 days after AFNT-212 infusion. When subjects are in the clinic, temperatures will be taken / recorded by study staff. When not in the clinic, temperatures will be taken by the subject or their personal caregiver and recorded in a temperature diary. f. During the DLT period, on days when the subject does not visit the study site, the subject's personal caregiver will be required to complete the ICE assessment. g. On the day of AFNT-212 infusion (D1), vital signs (including O2 saturation) should be obtained prior to the start of the infusion, every 15 minutes during the infusion, within 5 minutes after the end of the infusion, and approximately every hour for 2 hours after the end of the infusion. h. MUGA / ECHO and / or pulmonary function testing is required if indicated (i.e., history of pulmonary dysfunction or cardiovascular abnormalities). For subjects who have received potentially cardiotoxic bridging therapy, a repeat ECHO or MUGA is required within 2 weeks of the LDC. i. Serum or urine pregnancy test for SOCBP within 72 hours prior to planned leukapheresis. A serum pregnancy test is also required within 72 hours prior to LDC. j. Standard of care laboratory tests, if performed in accordance with protocol requirements, may be used to assess subject eligibility if collected within the eligibility screening period. Blood draws for laboratory tests do not need to be repeated. k. Clinically relevant CEA, CA-125, CA-19-9, CA-242 according to SOC. l. Inflammatory markers are assessed when clinically indicated (eg, when the subject develops a fever or symptoms of CRS or TLS). m. If the time between the screening assessment and leukapheresis is more than 7 days, the relevant screening assessment must be repeated and within the defined range prior to leukapheresis. n. Brain MRI is required at screening. Disease will be assessed according to RECIST 1.1 based on CT scans with contrast, including the chest, abdomen, and pelvis. For subjects / study sites unable to undergo / access CT scans, MRI or PET / CT scans may be used. The same modality used for imaging at baseline should be used at each subsequent time point. Scans will be collected for review by a central imaging vendor. o. Standard of care images taken prior to study entry may be used to determine subject eligibility for this study. Standard of care images used to assess eligibility must be obtained within 45 days of leukapheresis. p. Baseline imaging must be within 28 days prior to the start of the first planned AFNT-212 infusion. Subjects must be re-imaged if undergoing bridging therapy. q. Imaging is acquired until M24 / PTFD or PD, whichever occurs first. During STFU, imaging is collected only if the subject has not had a PD event. Upon PTFD and / or documented PD, subjects will be asked to continue STFU / LTFU and will be followed for up to 15 years from the first AFNT-212 infusion. Subsequent anticancer therapy and response to each line of regimen will be collected. Subjects who do not agree to participate in STFU / LTFU will be followed every 3 months for vital status or through official records. s. If at any time the subject develops a fever or symptoms of toxicity, relevant evaluations may be performed. t. To confirm PD.

[0368] Table 13. Pharmacokinetic and biomarker activity schedule TIFF2026504480000045.tif246130 Abbreviations: AESI = adverse event of special interest; CRS = cytokine release syndrome; D = days; ctDNA = circulating tumor DNA; DLT = dose-limiting toxicity; EoT = end of treatment; KRAS = Kirsten rat sarcoma viral oncogene homolog; LDC = lymphocyte-depleting chemotherapy; LTFU = long-term follow-up; M = months; PD = progressive disease; PK = pharmacokinetics; PTFD = post-treatment follow-up discontinued; RCL = replication-competent lentivirus; scRNAseq = single-cell RNA sequencing; follow-up; TCR = T-cell receptor; TLS = tumor lysis syndrome a. Consult the laboratory manual. If any tumor is not amenable to biopsy or if biopsy is not safe and / or feasible, request it at a subsequent visit. b. One core or equivalent of the baseline biopsy should be reserved for development of potential KRAS diagnostics. c. The initial research biopsy should be supplemented with a blood sample or alternatively a buccal swab for a normal tissue control. d. If the subject presents with an AESI or if such testing is clinically indicated or clinically appropriate, an evaluation may be performed at an additional time. e. Blood for molecular PK (persistence), cellular PK, and cytokine levels will be collected pre-dose and 120 minutes (± 30 minutes) post-dose (i.e., after the end of the infusion). Persistence (molecular PK) tests will be collected at 3, 6, 9, and 12 months, then every 6 months for up to 5 years. If persistence is demonstrated after 5 years, tests will be performed annually until undetectable. If all samples are negative during the first year, remaining samples can be stored. f. If a necropsy is performed in the event of subject death, and death occurs during the STFU and LTFU observation periods, blood and tissue samples should be requested to potentially test for vector persistence, transgene expression, and other relevant analyses at the molecular, cellular, or tissue level. g. Serum collected during the LDC period may be used for pharmacokinetic evaluation of fludarabine and cyclophosphamide, and / or other molecular species. h. Blood collected for immune profiling may be used to assess the frequency and / or phenotype of cell populations in the periphery.

[0369] Example 16: Large-scale manufacturing process for KRAS G12D TCR T cells Peripheral blood mononuclear cells (PBMCs) were first isolated. The resulting isolates were enriched for CD4 / CD8+ cells using a CliniMACS. The enriched isolates were then transferred to a sterile culture system (GRex 500M) for activation.

[0370] Cells were activated in an activation buffer containing TGF-β, human platelet lysate, and cytokines (eg, IL-2, IL-7, IL-15, IL-21).

[0371] Next, DNA for knock-in was prepared (Nanoplasmid® construct containing the sequence encoding KRAS G12D TCR), and the activated cells were contacted with DNA during electroporation (Xenon) for knock-in using homology-directed repair (HDR). Briefly, electroporation was used to transduce activated cells with sgRNA, nuclease, and DNA. Once inside the cells, sgRNA was used to guide DNA to the TRAC site for knock-in using MG29-1 nuclease.

[0372] Electroporation was performed in an isotonic electroporation buffer. Multiple sets of electroporations (e.g., four sets) were performed, each followed by an extended rest period (e.g., 10-40 min or longer). After electroporation, cells were transferred to a culture vessel for expansion in expansion buffer containing human platelet lysate.

[0373] Eight days after activation began, cells were fed with additional cytokines (eg, IL-2, IL-7, IL-15, IL-21).

[0374] Cells were then harvested for storage or cryopreservation.

[0375] The results showed that there were significant advantages to using the above process. Without intending to be bound by theory, the addition of TGF-β significantly increased the expression of various classical tissue residency (T) proteins, including CD103 and CD39, compared to a buffer lacking TGF-β. RM ) markers were found to be favorably upregulated, and the addition of human platelet lysate during the activation and expansion phases was shown to improve cell growth and yield compared to buffers lacking human platelet lysate. The use of an isotonic electroporation buffer during electroporation was shown to significantly increase both knock-in frequency and expansion potential compared to the use of standard electroporation buffer (Thermo Fisher). A prolonged post-electroporation rest period was shown to significantly increase knock-in efficiency and the final yield of transduced T cells. The timing of the cytokine feeding regimen was also shown to significantly improve knock-in efficiency and cell yield.

[0376] Other Aspects While embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the appended claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby. All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. a heterologous extracellular binding protein capable of binding to a peptide:HLA complex, wherein the peptide comprises a KRAS G12D mutant peptide; and Genomic mutations that decrease the expression of endogenous T-cell receptor alpha constant (TRAC), T-cell receptor beta constant 1 (TRBC1), or T-cell receptor beta constant 2 (TRBC2) A host cell comprising:

2. The host cell of claim 1, wherein the peptide:HLA complex comprises an HLA protein encoded by the HLA-A*11 allele.

3. The host cell of claim 2, wherein the peptide:HLA complex comprises an HLA protein encoded by the HLA-A*11:01 allele.

4. 3. The host cell of claim 1 or 2, wherein the extracellular binding protein comprises a T cell receptor (TCR) alpha chain variable (Vα) region, a TCR beta chain variable (Vβ) region, a T cell receptor (TCR) alpha chain constant (Cα) region, or a T cell receptor (TCR) beta chain constant (Cβ) region.

5. The host cell of claim 1, wherein the KRAS G12D mutant peptide comprises the amino acid sequence VVVGADGVGK.

6. The host cell of claim 5, wherein the Vα domain or the Vβ domain is human, humanized, or chimeric.

7. 7. The host cell of any one of claims 1 to 6, wherein the extracellular binding protein is human, humanized, or chimeric.

8. The extracellular binding protein is a TCR alpha chain variable (Vα) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 123, 139, 155, 171, 187, 229, 230, 239, 240, 249, 250, 259, 260, 269, and 270; a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 131, 147, 163, 179, 195, 234, 235, 244, 245, 254, 255, 264, 265, 274, and 275; or SEQ ID NO: 2-8, 10-16, 18-24, 26-32, 34-40, 42-48, 50-56, 58-64, 66-72, 74-80, 82-88, 90-96, 98 ~104, 106~112, 124~130, 132~138, 140~146, 148~154, 156~162, 164~170, 172~178, 180 -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278. A TCRα FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a TCRβ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, comprising an amino acid sequence having at least 80% sequence identity to any one of -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278.

8. The host cell of any one of claims 1 to 7, comprising:

9. The host cell of any one of claims 1 to 8, wherein the extracellular binding protein specifically binds to the KRAS G12D mutant peptide.

10. 10. The host cell of claim 9, wherein the extracellular binding protein is at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold selective for the KRAS G12D mutant peptide over other 10-mer peptides encoded by the genome of the cell.

11. 11. The host cell of any one of claims 1 to 10, further comprising (i) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain, or (ii) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain.

12. the KRAS extracellular binding protein is about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less Log for G12 mutant peptide 10 12. The host cell of any one of claims 1 to 11, having an EC50.

13. When the host cell is in the presence of tumor cells expressing the KRAS G12D mutant peptide, CD137 expression on the host cell is (i) CD137 expression by a reference human T cell that does not express the binding protein when the reference human T cell is in the presence of the tumor cell; or (ii) CD137 expression by the human T cells expressing the binding protein in the absence of the tumor cells or in the absence of antigen-presenting cells expressing the peptide:HLA complex. The host cell of any one of claims 1 to 12, wherein the expression level is increased compared to

14. The host cell of any one of claims 1 to 13, wherein the genomic mutation causing or contributing to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2) comprises an indel in the TRAC, TRBC1, or TRBC2 locus.

15. The host cell of any one of claims 1 to 14, wherein the genomic mutation that causes or contributes to reduced expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2) is a missense mutation that causes or contributes to reduced function or stability of a T cell receptor alpha or T cell receptor beta polypeptide encoded by the genome of the cell.

16. The host cell of any one of claims 1 to 14, wherein the genomic mutation that reduces expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2) results in premature termination of the T cell receptor alpha or T cell receptor beta polypeptide encoded by the endogenous TRAC, TRBC1, or TRBC2.

17. The host cell of any one of claims 1 to 16, comprising a genomic mutation that reduces expression of both: (i) TRAC; and (ii) TRBC1 or TRBC2.

18. The host cell of claim 17, comprising a genomic mutation that reduces expression of TRAC, TRBC1, and TRBC2.

19. 19. The host cell of any one of claims 1 to 18, comprising an immune cell or a precursor thereof.

20. 20. The host cell of claim 19, wherein the immune cells comprise T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof.

21. The immune cells include T cells, and the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - 20. The host cell of claim 19, comprising double-negative T cells, γδ T cells, or any combination thereof.

22. A polynucleotide encoding a heterologous extracellular binding protein inserted into the TRAC, TRBC1, or TRBC2 gene locus. A host cell comprising: the extracellular binding protein is capable of binding to a peptide:HLA complex, the peptide comprises a KRAS G12D mutant peptide, and the host cell has reduced expression of TRAC, TRBC1, or TRBC2. The host cell.

23. 23. The host cell of claim 22, wherein the polynucleotide encoding a heterologous extracellular binding protein is inserted into the TRAC locus and the host cell has reduced expression of TRAC.

24. 24. The host cell of any one of claims 1 to 23, further comprising a recombinant protein comprising the IL-7 receptor alpha (IL7RA) intracellular domain and the IL7RA transmembrane domain.

25. The host cell of claim 24, wherein the IL7RA intracellular domain has at least 80% sequence identity to SEQ ID NO:

224.

26. 26. The host cell of claim 24 or 25, wherein the IL7RA transmembrane domain has at least 80% sequence identity to SEQ ID NO:

225.

27. The host cell of any one of claims 24 to 26, wherein the recombinant protein further comprises a CD34 extracellular domain or a CD58 extracellular domain.

28. 28. The host cell of claim 27, wherein the CD58 extracellular domain has at least 80% sequence identity to SEQ ID NO:

227.

29. 29. The host cell of any one of claims 24 to 28, wherein the recombinant protein has at least 80% sequence identity to SEQ ID NO:

223.

30. A polynucleotide encoding an extracellular binding protein, the extracellular binding protein comprising: a TCR alpha chain variable (Vα) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 123, 139, 155, 171, 187, 229, 230, 239, 240, 249, 250, 259, 260, 269, and 270; a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 131, 147, 163, 179, 195, 234, 235, 244, 245, 254, 255, 264, 265, 274, and 275; or SEQ ID NO: 2-8, 10-16, 18-24, 26-32, 34-40, 42-48, 50-56, 58-64, 66-72, 74-80, 82-88, 90-96, 98 ~104, 106~112, 124~130, 132~138, 140~146, 148~154, 156~162, 164~170, 172~178, 180 -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278. A TCRα FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a TCRβ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, comprising an amino acid sequence having at least 80% sequence identity to any one of -186, 188-194, 196-202, 231-233, 236-238, 241-243, 246-248, 251-253, 25-258, 261-263, 266-268, 271-273, and 276-278. The polynucleotide comprising:

31. 31. The polynucleotide of claim 30, wherein the binding protein is capable of binding to a peptide:HLA complex, and the peptide comprises a KRAS G12 mutant peptide.

32. 32. The polynucleotide of claim 30 or 31, wherein the KRAS G12 mutant peptide is a KRAS G12D mutant peptide.

33. 33. The polynucleotide of claim 32, wherein the KRAS G12D mutant peptide comprises the amino acid sequence VVVGADGVGK.

34. 34. The polynucleotide of any one of claims 30 to 33, wherein the nucleic acid sequence is codon optimized.

35. 35. The polynucleotide of any one of claims 30 to 34, wherein the extracellular binding protein is human, humanized, or chimeric.

36. 36. The polynucleotide of any one of claims 30 to 35, wherein the extracellular binding protein is selective for the KRAS G12D mutant peptide.

37. the KRAS extracellular binding protein is about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, or about -9.2 or less Log for G12 mutant peptide 10 37. The polynucleotide of any one of claims 31 to 36, having an EC50.

38. 38. The polynucleotide of any one of claims 30-37, wherein the extracellular binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 229, 230, 234, 235, 239, 240, 244, 245, 249, 250, 254, 255, 259, 260, 264, 269, 265, 270, 274, and 275.

39. 39. The polynucleotide of any one of claims 30 to 38, further comprising a promoter.

40. 40. The polynucleotide of claim 39, wherein the promoter is the elongation factor-1 alpha (EF-1α) promoter.

41. 39. The polynucleotide of any one of claims 30 to 38, comprising RNA, DNA, or a combination thereof.

42. A vector comprising the polynucleotide of any one of claims 30 to 41.

43. 43. The vector of claim 42, which is a lentiviral vector, a gamma-retroviral vector, or an adeno-associated viral (AAV) vector.

44. A cell comprising the polynucleotide of any one of claims 30 to 41, or the vector of claim 34 or 35.

45. 30. A pharmaceutical composition comprising the host cell of any one of claims 1 to 29 and a pharmaceutically acceptable carrier, excipient, or diluent.

46. 46. ​​The pharmaceutical composition of claim 45, comprising both CD4+ cells and CD8+ cells having (i) the extracellular binding protein and (ii) the genomic mutation that reduces expression of the endogenous T cell receptor alpha constant (TRAC), T cell receptor beta constant 1 (TRBC1), or T cell receptor beta constant 2 (TRBC2).

47. 47. The pharmaceutical composition of claim 46, further comprising either or both of: (i) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha (CD8α) chain; or (ii) a transgenic polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta (CD8β) chain.

48. 48. The pharmaceutical composition of any one of claims 45-47, comprising about a 1:1 ratio of CD4+ T cells to CD8+ T cells.

49. 10. A method of treating a disease or disorder associated with a KRAS G12 mutation in a subject, the method comprising administering to the subject an effective amount of a host cell of any one of claims 1 to 29 or a pharmaceutical composition of any one of claims 45 to 48.

50. 50. The method of claim 49, wherein the subject is positive for the HLA-A*11 allele.

51. 50. The method of claim 49, wherein the subject is positive for the HLA-A*11:01 allele.

52. The method of any one of claims 49 to 51, wherein the KRAS G12 mutation is a KRAS G12D mutation.

53. 53. The method of any one of claims 49 to 52, wherein the disease or disorder comprises cancer.

54. 54. The method of claim 53, wherein the cancer is a solid cancer.

55. 54. The method of claim 53, wherein the cancer is a hematological malignancy.

56. The disease or disorder is selected from the group consisting of bile duct tumors, cholangiocarcinoma, colon adenocarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC); colorectal cancer; lung cancer, non-small cell lung cancer; biliary tract cancer; endometrial cancer; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, myeloid leukemia, acute myeloid leukemia; myelodysplastic syndrome; lymphoma, non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); cancer of the urinary tract; cancer of the small intestine; breast cancer; melanoma, cutaneous melanoma, anal melanoma, or mucosal melanoma; glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; and bladder cancer.

56. The method of any one of claims 53-55, wherein the tumor is selected from: sarcoma; glioblastoma; lung squamous cell carcinoma; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

57. The method of any one of claims 49 to 56, wherein the subject is determined to carry a KRAS G12D allele prior to said administering step.

58. 58. The method of any one of claims 49-57, wherein said subject has been genotyped for an HLA-A allele prior to said administering step.

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