Modified cytotoxic T cells and methods of use thereof

Abstract

The present disclosure provides modified cytotoxic T cells (mCTLs), the mCTLs comprising: a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide presenting a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); and b) one or more nucleic acids comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for a cancer-associated antigen. The present disclosure provides methods of generating the mCTLs. The present disclosure provides methods of treating cancer, comprising administering the mCTLs to an individual in need thereof.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 336,041, filed April 28, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The Sequence Listing is provided herein as Sequence Listing XML "CUEB-150WO_SEQ_LIST" having a size of 136,686 bytes, created on April 23, 2023. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] Introduction Cell therapy products include T cells engineered to express proteins that bind to proteins on cancer cells. For example, "CAR-T cells" contain a chimeric antigen receptor ("CAR") that targets a tumor-associated antigen. CAR-T cells can kill tumor cells and have been used successfully to treat cancer. For example, CAR-T cells targeting CD19, BCMA, CD30, CD22, or CD20 have shown significant activity in clinical trials. However, CAR-T therapy can suffer from drawbacks. Due to the large number of CAR-T cells administered, patients typically require lymphodepleting chemotherapy regimens prior to CAR-T cell administration. In addition, to maintain therapeutic levels of CAR-T cells after administration, patients may also require treatment with aldesleukin (Proleukin®), which can result in severe adverse side effects. Additionally, some patients may not achieve the desired or optimal results due to suboptimal proliferation or persistence of CAR-T cells in vivo. Summary of the Invention

[0004] overview The present disclosure includes disclosure of modified cytotoxic T cells (mCTLs), the mCTLs comprising: a) Amino acid sequence YMLDLQPETT (SEQ ID NO: 1) ("HPV E7 11-20 TCR") or YMLDLQPET (SEQ ID NO:2) ("HPV E7 11-19 TCR") ("HPV E7 11-19 / 20 The mCTLs may include a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide that presents a human papillomavirus (HPV) E7 peptide, the T cell receptor (TCR) comprising a nucleotide sequence encoding a T cell ... 11-19 / 20 This includes disclosure of mCTLs with TCRs other than TCR, as well as disclosure of T cells engineered to present both a TCR and a CAR or other cancer associated antigen binding domain, e.g., engineered cells other than NK cells. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 is a schematic diagram of the generation and use of mCTLs according to the present disclosure. [Diagram 2] 2A-2D provide exemplary TCR alpha and beta chain amino acid sequences (SEQ ID NOs:19-22). [Diagram 3] Figures 3A-3D provide the amino acid sequence of the T cell modulatory polypeptide (TMP) polypeptide (SEQ ID NO: 15) (Figure 3A), as well as the amino acid sequences of components of the polypeptide (SEQ ID NOs: 5, 8, and 10) (Figures 3B-3D). [Figure 4] 4A-4B provide the amino acid sequence of a TM polypeptide (SEQ ID NO:14) (FIG. 4A) and a β2M polypeptide (SEQ ID NO:4) (FIG. 4B). [Figure 5-1]FIG. 5 provides sequence identifiers for certain amino acid sequences (SEQ ID NOs: 1-18) annotated in the specification and drawings. [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified non-natural, or derivatized nucleotide bases.

[0007] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0008] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when the two sequences are compared and aligned, that percentage of bases or amino acids are the same and in the same relative positions. Sequence identity can be measured using a number of different methods. To determine sequence identity, sequences may be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.

[0009] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine, the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine, the group of amino acids with amide-containing side chains consists of asparagine and glutamine, the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan, the group of amino acids with basic side chains consists of lysine, arginine, and histidine, the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid, and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0010] "T cells" are helper T cells (CD4+ cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including T cell regulators (TCR), regulatory T cells (Treg), and NK-T cells.

[0011] The terms "expression construct" or "DNA construct" are used interchangeably herein and refer to a DNA molecule comprising a vector and at least one insert.

[0012] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and is also referred to as the dissociation constant (K D ). The affinity may be at least 1-fold, 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 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, or at least 1,000-fold, or more, of the antibody affinity for an unrelated amino acid sequence. The affinity of an antibody for a target protein may be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments. Unless otherwise indicated herein, when the word "about" is used in connection with a numerical value, it means a range of ±10% of the stated numerical value, e.g., "about 10" means a value between 9 and 11.

[0013] The term "binding" as used herein (e.g., with respect to binding of a T cell modulating polypeptide ("TMP") to a polypeptide on a T cell (e.g., a T cell receptor) or with respect to binding of an antigen-binding polypeptide present in a CAR to an antigen, such as a cancer-associated antigen) refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, e.g., by electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Non-covalent interactions typically occur over a 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than M or 10 -15 Dissociation constant (K D "Affinity" refers to the strength of non-covalent binding, with high binding affinity being characterized by a low K D "Specific binding" generally correlates with at least about 10 -7 M or more, e.g., 5×10 -7 M, 10 -8 M, 5×10 -8 M, 10 -9 "Non-specific binding" refers to binding with an affinity of at least about 10 -7 Binding with an affinity of less than M (e.g., 10 -6 M, 10 -5 M, 10 -4 "Specific binding" refers to binding with an affinity of at least one M (e.g., binding of a ligand to a moiety other than its designated binding site or receptor). However, in some contexts, e.g., binding between a TCR and a peptide / MHC complex, "specific binding" may be in the range of 1 μM to 100 μM, or 100 μM to 1 mM. As used herein, "covalently binding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two distinct molecules.

[0014] As used herein, the terms "treatment", "treating" and the like generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject susceptible to, but not yet diagnosed as having, the disease or condition, (b) inhibiting the disease or condition, i.e., arresting its onset, and / or (c) relieving the disease, i.e., regressing the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest are treatments of ongoing diseases that stabilize or reduce the undesirable clinical symptoms of the patient. Such treatments are desirably performed before complete loss of function of the affected tissue. The therapy will desirably be administered during, and in some cases after, the symptomatic stage of the disease.

[0015] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), etc.

[0016] As used herein, the term "about" used in connection with an amount indicates that the amount may vary by 10% of the amount stated. For example, "about 100" means an amount of 90 to 110. When about is used in the context of a range, "about" used in connection with a lower amount in a range means that the lower amount includes an amount 10% lower than the lower amount in the range, and "about" used in connection with a higher amount in a range means that the higher amount includes an amount 10% higher than the upper amount in the range. For example, about 100 to about 1000 means that the range extends from 90 to 1100.

[0017] Unless otherwise indicated, the term "substantially" is intended to encompass both "entirely" and "largely, but not entirely." For example, an Ig Fc that "does not substantially induce ADCC" means an Ig Fc that does not induce ADCC at all or that induces very little ADCC. As used herein, unless otherwise indicated, the term "substantially" means "entirely or largely, but not entirely." As another example, a "substantially homogenous population" means a population that is homogenous throughout or that is largely, but not entirely, homogenous.

[0018] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins that include an antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, such as, for example, biotin (a member of the biotin-avidin specific binding pair). The terms also encompass Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by the same group of cells, all of which were produced from a single cell by repeated cell replication. That is, a clone of cells produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains, two heavy chains and two light chains connected by disulfide bonds.

[0019] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin that contains portions of immunoglobulins of different origins, at least one portion of which contains an amino acid sequence of human origin. For example, a humanized antibody can contain portions derived from immunoglobulins of non-human origin with the requisite specificity, e.g., mouse, and immunoglobulin sequences of human origin (e.g., chimeric immunoglobulins), portions that are chemically combined by conventional techniques (e.g., synthesis), or portions that are prepared as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of a chimeric antibody can be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin that contains one or more immunoglobulin chains that contain complementarity determining regions (CDRs) derived from an antibody of non-human origin and framework regions derived from light and / or heavy chains of human origin (e.g., CDR-grafted antibodies, with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., U.S. Patent No. 4,816,567, European Patent No. 0,125,023 B1, U.S. Patent No. 4,816,397, European Patent No. 0,120,694 B1, International Publication No. WO 86 / 01533, European Patent No. 0,194,276 B1, U.S. Patent No. 5,225,539, European Patent No. 0,239,400 B1, and European Patent Application Publication No. 0,519,596 A1. See also U.S. Patent No. 4,946,778, U.S. Patent No. 5,476,786, and Bird et al. (1988) Science 242:423, regarding single chain antibodies.

[0020] The term "nanobody" (Nb) as used herein refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH), known to those skilled in the art. They are derived from heavy chain-only antibodies found in the Camelidae family (see Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (1996) Nature Structural Biol. 3:803; and Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). In the "Camelidae" family, immunoglobulins are found that lack light polypeptide chains. "Camelidae" includes Old World Camelidae (Camelus bactrianus and Camelus dromedarius) and New World Camelidae (e.g., Alpaca (Llama paccos), Llama glama, Guanaco (Llama guanicoe), and Vicuña (Llama vicugna). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH These are called antibodies.

[0021] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0022] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0023] "Fab" fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0024] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0025] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V H and VL domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0026] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L Diabodies comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) within a 5'-terminal linker. By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0027] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al (1977) J. Biol. Chem. 252:6609, Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987), and MacCallum et al. (1996) J. Mol. Biol. 262:732, and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs as defined by each of the above cited documents are set forth in Table 1 below for comparison.

[0028] [Table 1]

[0029] As used herein, the terms "CDR-L1", "CDR-L2", and "CDR-L3" refer to the first, second, and third CDRs, respectively, in a light chain variable region. As used herein, the terms "CDR-H1", "CDR-H2", and "CDR-H3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region. As used herein, the terms "CDR-1", "CDR-2", and "CDR-3" refer to the first, second, and third CDRs, respectively, in the variable region of either chain.

[0030] As used herein, the term "framework", when used in reference to an antibody variable region, is intended to mean all amino acid residues outside the CDR regions in the variable region of an antibody. Variable region frameworks are generally discontinuous amino acid sequences about 100-120 amino acids in length, but are intended to refer only to those amino acids outside the CDRs. As used herein, the term "framework region" is intended to mean each domain of the framework separated by the CDRs.

[0031] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0032] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may be independently included in the narrower range, and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. Where an explicit range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the disclosure.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which they are cited.

[0034] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "modified T cells" includes a plurality of such T cells, a reference to a "T cell modulating polypeptide" or "TMP" includes a reference to one or more T cell modulating polypeptides or TMPs and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0035] It is to be understood that certain features of the present disclosure that are described in the context of individual embodiments for clarity may also be provided in combination within a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if all combinations were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and expressly disclosed herein.

[0036] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0037] Detailed Description The present disclosure provides modified cytotoxic T cells (mCTLs), the mCTLs comprising: a) an antibody having the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) ("HPV E7 11-20 TCR") or YMLDLQPET (SEQ ID NO:2) ("HPV E7 11-19 TCR") ("HPV E7 11-19 / 20The mCTLs may include a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide that presents a human papillomavirus (HPV) E7 peptide, the T cell receptor (TCR) comprising a nucleotide sequence encoding a T cell ... 11-19 / 20 This includes disclosure of mCTLs with TCRs other than TCR, as well as disclosure of T cells engineered to present both a TCR and a CAR or other cancer associated antigen binding domain, e.g., engineered cells other than NK cells.

[0038] Engineered Cytotoxic T Cells The present disclosure provides modified cytotoxic T cells (mCTLs), which comprise: a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide that presents a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); and b) one or more nucleic acids comprising a nucleotide sequence encoding an antigen-binding polypeptide, such as a CAR or other polypeptide comprising an antigen-binding domain specific for a cancer-associated antigen.

[0039] A T cell receptor (TCR) specific for an MHC class I polypeptide presenting a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) is referred to herein as "HPV E7 11-19 / 20 It is called "TCR".

[0040] This disclosure relates to HPV E7 11-19 / 20 The present disclosure provides a method for generating mCTLs comprising a TCR. 11-19 / 20The present disclosure also provides a method for treating cancer comprising administering to an individual in need thereof an mCTL comprising a TCR. 11-19 / 20 Provided are mCTLs that comprise a TCR and an antigen-binding polypeptide, such as a CAR or other polypeptide that comprises an antigen-binding domain specific for a cancer-associated antigen.

[0041] Thus, the present disclosure provides: a) HPV E7 11-19 / 20 The present invention provides an mCTL comprising: a) one or more nucleic acids comprising a nucleotide sequence encoding a TCR; and b) one or more nucleic acids comprising a nucleotide sequence encoding a CAR comprising an antigen-binding domain specific for a cancer-associated antigen.

[0042] Thus, the present disclosure provides: a) HPV E7 11-19 / 2011-19 / 20 The present invention provides an mCTL comprising a) a TCR and b) a CAR comprising an antigen-binding domain specific for a cancer-associated antigen.

[0043] As used herein, mCTL refers to HPV E7 11-19 / 20Although exemplified as having a T cell receptor (TCR) specific for an MHC class I polypeptide presenting a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), it will be understood that the mCTL may have an alternative TCR specific for a different peptide, in which case a TMP presenting the peptide for which the alternative TCR is specific is used. Similarly, although the mCTL is exemplified as comprising a CAR, the mCTL may be other cell therapy products that comprise an antigen binding domain for a cancer-associated antigen other than a CAR. mCTLs that comprise a TCR specific for a different peptide and / or an antigen binding domain other than a CAR are expressly contemplated by the present disclosure. Similarly, although the present disclosure is primarily directed to modified cytotoxic T cells (mCTLs), such as CAR-T cells, it will be understood that the disclosure herein can be used to modify other types of cytotoxic cells, e.g., NK cells, macrophages, ILCs (innate lymphoid cells), with natural or modified antigen binding domains. T cells can include classical T cells (alpha beta receptor expressing), or T cells expressing gamma / delta TCR, CD8 and CD4 phenotypes, MR-1 non-classical HLA-restricted MAIT T cells, killer T cells, and killer innate-like T cells.

[0044] In some examples, the mCTLs are in vitro. The in vitro compositions of the present disclosure can include a T cell population that may include T cells other than mCTLs. Such cells are referred to as "unmodified T cells." Thus, the in vitro compositions of the present disclosure can be a heterogeneous population that includes mCTLs and unmodified T cells. In some examples, 1% to 20% of the total number of T cells in the composition are mCTLs. In some examples, 1% to 5%, 5% to 10%, 10% to 15%, or 15% to 20% of the total number of T cells in the composition are mCTLs. In some examples, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% of the total number of T cells in the composition are mCTLs. In some examples, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the total number of T cells in the composition are mCTLs. Thus, in some examples, the population of T cells in the composition is a substantially homogenous population of mCTLs. As described above, a "substantially homogenous population" refers to a population that is homogenous throughout, or that is largely, but not entirely, homogenous.

[0045] An exemplary use of an in vivo composition comprising mCTL is shown generally in Figure 1. In vitro cell populations are engineered to express a CAR specific for a target cancer-associated antigen and to express a heterologous HPV E7 11-19 / 20 The targeted modified T cells are then administered to an individual in need thereof, for example, an individual with a cancer that expresses the targeted cancer-associated antigen. A T cell modulating polypeptide (TMP) comprising an HPV E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) can also be administered to the individual. The TMP binds to the HPV E7 peptide present in the TMP. 11-19 / 20The TMP comprises an immune modulating polypeptide that results in activation of T cells that contain a TCR. The modified target T cells (e.g., mCTLs) target cancer cells that express the cancer-associated antigen presented by the CAR. The TMP activates the modified target T cells by binding to the TCR present on the modified target T cells. Activation of the modified target can result in one or both of the following: (i) increased direct killing of cancer cells, and (ii) increased secretion of cytokines that promote the anti-cancer activity of resident immune cells. The TMP can increase the in vivo expansion of mCTLs, thereby reducing the number of mCTLs that need to be administered for anti-cancer therapy, and can reduce or substantially eliminate the need for lymphodepleting chemotherapy prior to administration of the mCTLs, and / or reduce or substantially eliminate the need for treatment with recombinantly produced IL-2, e.g., aldesleukin (Proleukin®), after administration of the CAR-T cells.

[0046] Chimeric Antigen Receptor As described above, the engineered T cells are engineered to express a CAR specific to a cancer-associated antigen. A CAR generally comprises: a) an extracellular domain comprising an antigen-binding domain (antigen-binding polypeptide), b) a transmembrane region, and c) a cytoplasmic domain comprising an intracellular signaling domain (intracellular signaling polypeptide). In some examples, a CAR comprises: a) an extracellular domain comprising an antigen-binding domain, b) a transmembrane region, and c) a cytoplasmic domain comprising i) one or more costimulatory polypeptides, and ii) an intracellular signaling domain. In some examples, a CAR comprises a hinge region between the extracellular antigen-binding domain and the transmembrane domain. Thus, in some examples, a CAR comprises: a) an extracellular domain comprising an antigen-binding domain, b) a hinge region, c) a transmembrane region, and d) a cytoplasmic domain comprising an intracellular signaling domain. In some examples, a CAR comprises: a) an extracellular domain comprising an antigen-binding domain, b) a hinge region, c) a transmembrane region, and d) a cytoplasmic domain comprising i) one or more costimulatory polypeptides, and ii) an intracellular signaling domain.

[0047] Exemplary CAR structures are known in the art (see, e.g., WO 2009 / 091826, U.S. Patent Application Publication No. 20130287748, WO 2015 / 142675, WO 2014 / 055657, WO 2015 / 090229, and U.S. Pat. No. 9,587,020).

[0048] In some examples, the CAR is a single polypeptide chain. In some examples, the CAR comprises two polypeptide chains. Generally, any CAR structure known to those skilled in the art can be used to modify T cells to prepare the compositions disclosed herein.

[0049] CARs specific to various tumor antigens, such as CD171-specific CAR (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CAR (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CAR (Kobold et al., J. Natl Cancer Inst (2014) 107(1):364), carbonic anhydrase IX-specific CAR (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), folate receptor-α (FR-α)-specific CAR (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CAR (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696, Nakzawa et al., Mol Ther (2011) 19(12):2133-2143, Ahmed at al., Mol Ther (2009) 17(10):1779-1787, Luo et al., Cell Res (2016) 26(7):850-853, Morgan et al., Mol Ther (2010) 18(4):843-851, Grada et al., Mol Ther Nucleic Acids (2013) 9(2):32), CEA-specific CAR (Katz et al., Clin Cancer Res (2015) 21(14):3149-3159), IL-13Rα2-specific CAR (Brown et al., Clin Cancer Res (2015) 21(18):4062-4072), ganglioside GD2-specific CAR (Louis et al., Blood (2011) 118(23):6050-6056, Caruana et al., Nat Med (2015) 21(5):524-529, Yu et al., (2018) J. Hematol. Oncol. 11:1), ErbB2-specific CAR (Wilkie et al., J Clin Immunol (2012) 32(5):1059-1070), VEGF-R-specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2): 154-166), mesothelin (MSLN)-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther(2015)23(10):1600-1610), CD19-specific CARs (axicabtagenecilloreucel (Yescarta™) and tisagenlecleucel (Kymriah™)) are known in the art. See also Li et al., J Hematol and Oncol (2018) 11:22; Heyman and Yan (2019) Cancers 11:pii:E191; Baybutt et al. (2019) Clin. Pharmacol. Ther. 105:71, which review clinical trials of tumor-specific CARs.

[0050] For example, CAR-T therapies include Yescarta® (axicabtagene ciloleucel), which is a CAR that contains an scFv that binds to CD19. As further examples, Tecartus® (brexucabtagene autoleucel) is a CAR that contains an scFv that binds to CD19, Kymriah® (tisagenleucleucel) is a CAR that contains an scFv that binds to CD19, Abecma® (idecabtagene vicleucel) is a CAR that contains an scFv that binds to BCMA, and Breyanzi® (lisocabtagene maraleucel) is a CAR that contains an scFv that binds to CD19.

[0051] Antigen-binding domain As mentioned above, the CAR comprises an extracellular domain that comprises an antigen-binding domain. The antigen-binding domain present in the CAR can be any antigen-binding polypeptide, and a wide variety of antigen-binding polypeptides are known in the art. In some examples, the antigen-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelidae antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains are suitable. In some examples, the antigen-binding domain is a nanobody.

[0052] In some examples, the antigen bound by the antigen binding domain of the CAR is a MUC1 polypeptide, an LMP2 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2 / neu polypeptide, a melanoma antigen family A, 3 (MAGE A3) polypeptide, a p53 polypeptide, a mutant p53 polypeptide, a NY-ESO-1 polypeptide, a folate hydrolase (prostate specific membrane antigen; PSMA) polypeptide, a carcinoembryonic antigen (CEA) polypeptide, a melanoma antigen recognized by T cells (melanA / MART1) polypeptide, a Ras polypeptide, a gp100 polypeptide, a proteinase 3 (PR1) polypeptide, a bcr-abl polypeptide, a tyrosinase polypeptide, a survivin polypeptide, a prostate specific antigen (PSA) polypeptide, an hTERT polypeptide, a sarcoma metastasis breakpoint polypeptide, a synovial sarcoma X (SSX) breakpoint polypeptide, an EphA2 polypeptide, an acid phosphatase, prostate (PAP) polypeptide, a melanoma inhibitor of apoptosis (ML-IAP) polypeptide, an epithelial cell adhesion molecule (EpCAM) polypeptide, an ERG (TMPRSS2 ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma associated antigen-1 (MAGE A1) polypeptide, cytochrome P4501B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma cell antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen, also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high molecular weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase with Ig and EGF homology domains-2 (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, or Fos-related antigen-1 (FOSL) polypeptide. In some examples, the antigen is a human papillomavirus (HPV) antigen. In some examples, the antigen is an alpha-fetoprotein (AFP) antigen. In some examples, the antigen is a Wilms Tumor-1 (WT1) antigen.

[0053] The antigen-binding polypeptide of the CAR can bind to any of a variety of cancer-associated antigens, including, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), B-cell maturation antigen (BCMA), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Furthermore, examples of cancer-related antigens include 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein (AFP), BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-I receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0054] In some examples, the cancer-associated antigen bound by the antigen-binding polypeptide of the CAR is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2. In some examples, the cancer-associated antigen is BCMA. In some examples, the cancer-associated antigen is MUC1. In some examples, the cancer associated antigen is CD 19. In some examples, the cancer associated antigen is AFP.

[0055] The VH and VL amino acid sequences of various cancer-associated antigen binding antibodies are known in the art, as are the light and heavy chain CDRs of such antibodies.See, for example, Ling et al. (2018) Frontiers Immunol. 9:469, International Publication No. 2005 / 012493, US Patent Application Publication No. 2019 / 0119375, US Patent Application Publication No. 2013 / 0066055.The following are non-limiting examples of antibodies that bind to cancer-associated antigens:

[0056] 1) Anti-Her2 In some instances, the anti-Her2 antibody a) the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25) and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26) In some embodiments, the heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0057] In some examples, the anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody comprises a) the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 27) and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 28) The VH can comprise an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to In some examples, the anti-Her2 antibody is comprised, in order from N-terminus to C-terminus: a) the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 28) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) a linker, and c) the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 27) and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to: Suitable linkers are described elsewhere herein, and include, for example, (GGGGS)n (SEQ ID NO:29), where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0058] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0059] For example, an anti-Her2 antibody may comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 30), a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO: 31), a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO: 32), a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO: 33), a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO: 34), and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 35).

[0060] In some examples, the anti-Her2 antibody is an scFv antibody. For example, the anti-Her2 scFv has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 36) The amino acid sequence may include an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0061] As another example, in some instances, the anti-Her2 antibody a) the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 37) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38) The antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0062] In some examples, the anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody comprises a) the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 39) and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 40) The VH can comprise an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0063] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V LThe CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0064] For example, an anti-HER2 antibody may comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO: 41), a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO: 42), a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO: 43), a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO: 44), a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO: 45), and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 46).

[0065] In some examples, the anti-Her2 antibody is an scFv. For example, in some examples, the anti-Her2 scFv has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 36) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0066] 2) Anti-CD19 Anti-CD19 antibodies are known in the art, and the VH and VL, or VH and VL CDRs of any anti-CD19 antibody can be included in the CAR. See, e.g., WO 2005 / 012493.

[0067] In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 47), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 48), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 49). In some examples, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 50), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 51), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 52). In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 47), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 48), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 49), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 50), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 51), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 52).

[0068] In some examples, the anti-CD19 antibody is an scFv. For example, in some examples, the anti-CD19 scFv has the following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 53) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0069] 3) Anti-mesothelin Anti-mesothelin antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-mesothelin antibody can be included in a CAR. See U.S. Patent Application Publication No. 2019 / 0000944, WO 2009 / 045957, WO 2014 / 031476, U.S. Patent No. 8,460,660, U.S. Patent Application Publication No. 2013 / 0066055, and WO 2009 / 068204.

[0070] In some instances, the anti-mesothelin antibody a) the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO: 54) and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55) In some embodiments, the heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0071] In some examples, the anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-mesothelin antibody comprises a) the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO: 56) and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 57) The VH can comprise an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0072] In some examples, the anti-mesothelin antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0073] For example, an anti-mesothelin antibody may include a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO:58), a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO:59), a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO:60), a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO:61), a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO:62), and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO:63).

[0074] The anti-mesothelin antibody can be an scFv. As one non-limiting example, an anti-mesothelin scFv has the following amino acid sequence: TIFF2025515473000003.tif24162 (where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined) may include.

[0075] As one non-limiting example, an anti-mesothelin scFv can have the following amino acid sequence: TIFF2025515473000004.tif30160 (where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined) may include.

[0076] 4) Anti-BCMA Anti-BCMA (B cell maturation antigen) antibodies are known in the art, and the VH and VL, or VH and VL CDRs of any anti-BCMA antibody can be included in a CAR. See, e.g., WO 2014 / 089335, U.S. Patent No. 2019 / 0153061, and WO 2017 / 093942.

[0077] In some instances, the anti-BCMA antibody a) the amino acid sequence: QSVLTQPPSASGTPGPQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 66) and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67) In some embodiments, the heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0078] In some examples, the anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-BCMA antibody comprises a) the amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG (SEQ ID NO: 68) and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to b) the amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSV (SEQ ID NO: 69) The VH can comprise an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0079] In some examples, the anti-BCMA antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0080] For example, an anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO: 70), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO: 71), a VH CDR1 having the amino acid sequence GFTGFGDYA (SEQ ID NO: 72), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO: 73), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO: 74).

[0081] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv has the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 75) may include.

[0082] As another example, the anti-BCMA scFv may have the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 76) may include.

[0083] In some examples, the anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO: 77), a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO: 78), a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO: 79), a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO: 80), a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO: 81), and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO: 82).

[0084] In some instances, the anti-BCMA antibody a) the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 83) In some embodiments, the invention includes a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0085] In some instances, the anti-BCMA antibody a) the amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 84) In some embodiments, the heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0086] In some examples, the anti-BCMA antibody (e.g., the antibody referred to in the literature as belantamab) has the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 83) and a light chain comprising The amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 84) and a heavy chain comprising:

[0087] In some examples, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some examples, the anti-BCMA antibody is GSK2857916 (belantamab-mafodotin), and monomethyl auristatin F (MMAF) is linked to the anti-BCMA antibody belantamab via a maleimidocaproyl linker.

[0088] 5) Anti-MUC1 In some examples, the antigen-binding polypeptide present in the CAR is a single-chain Fv specific for MUC1.See, for example, Singh et al. (2007) Mol. Cancer Ther. 6:562, Thie et al. (2011) PLoSOne 6:e15921, Imai et al. (2004) Leukemia 18:676, Posey et al. (2016) Immunity 44:1444, EP 3130607, EP 3164418, WO 2002 / 044217, and US 2018 / 0112007.In some examples, the antigen-binding polypeptide present in the CAR is a scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:85). In some examples, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 86). In some examples, the antigen-binding polypeptide present in the CAR is an scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 87). In some examples, the TTP is an scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 88). In some examples, the antigen-binding polypeptide present in the CAR is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 89).

[0089] As an example, an anti-MUC1 antibody may comprise a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO: 90), a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO: 91), a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO: 92), a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO: 93), a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO: 94), and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO: 95). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0090] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 96), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO: 98), a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 99), a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO: 100), and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO: 101). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0091] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO: 102), a VH CDR2 having the amino acid sequence VISTFSGNINFNQKFKG (SEQ ID NO: 103), a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO: 104), a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 105), a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO: 106), and a VL CDR3 having the amino acid sequence (FQGSHVPFT (SEQ ID NO: 107). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0092] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO:96), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO:97), a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO:108), a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO:109), a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO:110), and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO:111). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0093] 6) Anti-MUC16 In some examples, the antigen-binding polypeptide present in CAR is specific to MUC16 polypeptide present on cancer cells.See, for example, US Patent Application Publication No. 2018 / 0118848 and US Patent Application Publication No. 2018 / 0112008.In some examples, the antigen-binding polypeptide specific to MUC16 is scFv.In some examples, the antigen-binding polypeptide specific to MUC16 is nanobody.

[0094] As an example, an anti-MUC16 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO: 112), a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO: 113), a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO: 114), a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO: 115), a VL CDR2 having the amino acid sequence TAS, and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO: 116). See, e.g., U.S. Patent Application Publication No. 2018 / 0118848.

[0095] 7) Examples of antigen-binding domains In some examples, a suitable CAR comprises an scFv specific for CD19. For example, in some examples, the anti-CD19 scFv has the following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 53) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0096] In some examples, a suitable CAR comprises an scFv specific for mesothelin. For example, in some examples, the anti-mesothelin scFv has the following amino acid sequence: QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGRYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATISCRASQSVSSNFAWYQQRPGQAPRLLIYDASNRATGIPPRFSGSGSGTDFTLTISSLEPED FAAYYCHQRSNWLYTFGQGTKVDIK (SEQ ID NO: 64) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0097] In some examples, the anti-mesothelin scFv has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRRTVVTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCQASQDISNSLNWYQQKAGKAPKLLIYDASTLETGVPSRFSGSGSGTDFSF TISSLQPEDIATYYCQQHDNLPLTFGQGTKVEIK (SEQ ID NO: 65) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0098] In some examples, a suitable CAR comprises an scFv specific for B cell maturation antigen (BCMA). For example, in some examples, an anti-BCMA scFv has the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 75) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0099] In some examples, the anti-BCMA scFv has the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 76) The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0100] Hinge Area As mentioned above, CAR may include a hinge region between the extracellular domain and the transmembrane domain. As used herein, the term "hinge region" refers to a flexible polypeptide connector region (also referred to herein as "hinge" or "spacer") that provides structural flexibility and spacing to adjacent polypeptide regions and may be composed of natural or synthetic polypeptides. The hinge region may include a complete hinge region derived from an antibody of a class or subclass different from that of the CH1 domain. The term "hinge region" may also include a region derived from CD8 and other receptors that perform similar functions of providing flexibility and spacing to adjacent regions.

[0101] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0102] As non-limiting examples, an immunoglobulin hinge region can comprise one of the following amino acid sequences: DKTHT (SEQ ID NO:117), CPPC (SEQ ID NO:118), CPEPKSCDTPPPCPR (SEQ ID NO:119), ELKTPLGDTTHT (SEQ ID NO:120), KSCDKTHTCP (SEQ ID NO:121), KCCVDCP (SEQ ID NO:122), KYGPPCP (SEQ ID NO:123), EPKSCDKTHTCPPCP (SEQ ID NO:124) (human IgG1 hinge), ERKCCVECPPCP (SEQ ID NO:125) (human IgG2 hinge), ELKTPLGDTTHTCPRCP (SEQ ID NO:126) (human IgG3 hinge), SPNMVPHAHHAQ (SEQ ID NO:127) (human IgG4 hinge), and the like. The hinge region can comprise an amino acid sequence derived from human CD8, for example, the hinge region can comprise the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 128), or a variant thereof.

[0103] Transmembrane domain Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic cell (e.g., mammalian) is suitable for use. The transmembrane region of the CAR may be any of the following: the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R.Alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 , CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A , Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp (i.e., including at least these transmembrane regions). The transmembrane domain may be synthetic, in which case it may contain primarily hydrophobic residues such as leucine and valine. In some instances, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0104] As one non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 129) can be used. Additional non-limiting examples of suitable TM sequences include: a) CD8 beta-derived TM: LGLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 130), b) CD4-derived TM: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 131), c) CD3 zeta-derived TM: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 132), d) CD28-derived TM: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 133), e) CD134 (OX40)-derived TM: VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 134), and f) CD7-derived TM: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 135).

[0105] Intracellular domain-costimulatory polypeptide The intracellular portion (cytoplasmic domain) of the CAR may comprise one or more costimulatory polypeptides. Non-limiting examples of suitable costimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. Suitable costimulatory polypeptides include, for example: 1) the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 136) 4-1BB polypeptides having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 2) the following amino acid sequence: FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 137) a CD28 polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 3) the amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 138) ICOS polypeptides having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 4) the amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 139) OX40 polypeptides having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 5) the amino acid sequence: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 140) a BTLA polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 6) the amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 141) a CD27 polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 7) the amino acid sequence: RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 142) a CD30 polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 8) the amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 143) and a GITR polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to 9) The amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 144) and HVEM polypeptides having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to HVEM. The costimulatory polypeptide can have a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa.

[0106] Intracellular domain - signaling polypeptide The intracellular portion of the CAR may comprise a signaling polypeptide. Suitable signaling polypeptides include, for example, immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2L / I, where X1 and X2 are, independently, any amino acid. In some examples, the intracellular signaling domain of the CAR comprises 1, 2, 3, 4, or 5 ITAM motifs. In some examples, the ITAM motif is repeated twice in the intracellular signaling domain, with the first and second ITAM motif instances separated from each other by 6-8 amino acids, e.g., (YX1X2L / I)(X3). n(YX1X2L / I), where n is an integer from 6 to 8, and each of the 6 to 8 X3 can be any amino acid. In some examples, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0107] Suitable intracellular signaling domains may be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains may be ITAM motif-containing domains derived from any ITAM motif-containing protein. Therefore, suitable intracellular signaling domains do not need to contain the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex binding protein alpha chain).

[0108] TCR As described above, the mCTLs of the present disclosure are capable of detecting HPV E7 11-19 / 20 The mCTL comprises (e.g., expressed on its surface) a TCR specific for the HPV E7 peptide YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) in complex with a class I MHC polypeptide (an MHC class I heavy chain polypeptide and a β2-microglobulin (β2M) polypeptide). The TCR is heterologous to the mCTL, i.e., the TCR is not naturally occurring in the mCTL.

[0109] In some examples, the mCTL comprises a heterologous TCR comprising (i) an alpha chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the D224 alpha chain amino acid sequence shown in Figure 2A, and (ii) a beta comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the D224 alpha chain amino acid sequence shown in Figure 2B. In some examples, the mCTL comprises a heterologous TCR comprising (i) an alpha chain having the amino acid sequence shown in Figure 2A, and (ii) a beta chain having the amino acid sequence shown in Figure 2B.

[0110] In some examples, the mCTL comprises a heterologous TCR comprising (i) an alpha chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the D224 alpha chain amino acid sequence shown in Figure 2C, and (ii) a beta comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the D224 alpha chain amino acid sequence shown in Figure 2D. In some examples, the mCTL comprises a heterologous TCR comprising (i) an alpha chain having the amino acid sequence shown in Figure 2C, and (ii) a beta chain having the amino acid sequence shown in Figure 2D.

[0111] Compositions Comprising mCTLs The present disclosure provides a composition comprising an mCTL of the present disclosure.The present disclosure provides a composition comprising T cells, wherein a percentage of the T cells that are mCTLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.

[0112] In addition to the mCTL of the present disclosure, the composition of the present disclosure may include one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizers; detergents, such as non-ionic detergents, such as Tween-20, etc.; protease inhibitors; glycerol, etc. The composition containing the mCTL may include a liquid medium in which the cells are maintained.

[0113] The composition may contain one or more pharma- ceutically acceptable excipients, the types of which are well known in the art and need not be discussed in detail herein.See, for example, Sheskey et al., "Handbook of Pharmaceutical Excipients" (2020), Vol. 9 (latest edition), and / or "Remington: The Science and Practice of Pharmacy", 23rd Ed. (2020), Vol. 23 (latest edition).

[0114] The pharmaceutical composition may comprise an amount of mCTL of the present disclosure and a pharma- ceutically acceptable excipient.In some examples, the pharmaceutical composition is suitable for administration to a subject, e.g., sterile.For example, in some examples, the pharmaceutical composition is suitable for administration to a human subject, e.g., when the composition is sterile and does not contain detectable pyrogens and / or other toxins, or when such detectable pyrogens and / or other toxins are present at levels within the acceptable limits set by applicable regulatory authorities, e.g., the United States Food and Drug Administration (USF&DA).

[0115] The present disclosure provides a container that contains the mCTLs (e.g., liquid compositions) of the present disclosure. The container can be, for example, a syringe, an ampoule, etc. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

[0116] In some examples, the mCTL is present in a liquid composition. Thus, the present disclosure provides a composition (e.g., a liquid composition (including a pharmaceutical composition)) comprising mCTL. In some examples, the composition of the present disclosure comprises a) mCTL and b) saline (e.g., 0.9% NaCl). In some examples, the composition is sterile. In some examples, the composition is suitable for administration to a human subject, for example, when the composition is sterile and free of detectable pyrogens and / or other toxins, or when such detectable pyrogens and / or other toxins are present at levels within acceptable limits set by applicable regulatory authorities, e.g., USF&DA. Thus, the present disclosure provides a composition comprising a) mCTL and b) saline (e.g., 0.9% NaCl), which is sterile and free of detectable pyrogens and / or other toxins, or when such detectable pyrogens and / or other toxins are present at levels within acceptable limits set by applicable regulatory authorities, e.g., USF&DA. In some examples, the composition further comprises TMP as described herein.

[0117] The present disclosure provides a mixture of the mCTL of the present disclosure and a TMP comprising an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1).The present disclosure provides a mixture of the mCTL of the present disclosure and a TMP comprising an HPV16 peptide comprising the amino acid sequence YMLDLQPET (SEQ ID NO: 2).

[0118] T-cell Modulatory Polypeptide (TMP) T cell modulating polypeptides (TMPs) suitable for use herein include either single chain or heterodimeric polypeptides comprising (i) a peptide, e.g., an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a β2 microglobulin (β2M) polypeptide; (iii) an immune modulating polypeptide (or "MOD"), such as an IL-2 polypeptide; (iv) a major histocompatibility complex class I (MHC class I) heavy chain polypeptide; and (v) a scaffolding polypeptide, such as an immunoglobulin (Ig) Fc polypeptide.

[0119] For example, the TMP can comprise a heterodimeric polypeptide comprising: a) a first polypeptide comprising: (i) an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1), (ii) a linker, and (iii) a β2 microglobulin (β2M) polypeptide; and b) a second polypeptide comprising: (i) two copies of an IL-2 polypeptide, (ii) a major histocompatibility complex class I (MHC class I) heavy chain polypeptide, and (iii) an immunoglobulin (Ig) Fc polypeptide, e.g., a variant IgG1 Fc polypeptide having a substantially reduced ability to effect complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0120] The β2M polypeptide has the following β2M amino acid sequence: In some examples, the β2M polypeptide can include an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025515473000005.tif10152. In some examples, the β2M polypeptide can include the following β2M amino acid sequence: TIFF2025515473000006.tif10152 (where amino acid 12 is Cys) In some examples, the β2M polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: Contains TIFF2025515473000007.tif10150.

[0121] In some examples, the IL-2 polypeptide is a variant of a naturally occurring costimulatory protein, which variant exhibits reduced affinity for its corresponding (cognate) costimulatory protein (e.g., IL-2R) on T cells compared to the affinity of the naturally occurring IL-2 polypeptide for the corresponding costimulatory protein (IL-2R). In some examples, the IL-2 variant exhibits reduced binding to IL-2Rα, thereby minimizing or substantially reducing activation of Tregs by the IL-2 variant. Alternatively, or additionally, in some examples, the IL-2 variant MOD exhibits reduced binding to IL-2Rβ, such that the IL-2 variant MOD exhibits reduced overall affinity for IL-2R. In some examples, the IL-2 variant MOD exhibits both properties, i.e., reduced or substantially no binding to IL-2Rα and reduced binding to IL-2Rβ, such that the IL-2 variant polypeptide exhibits reduced overall affinity for IL-2R. For example, IL-2 variants with substitutions at H16 and F42 showed reduced binding to IL-2Rα and IL-2Rβ. See Quayle et al., Clin Cancer Res; 26(8)April 15, 2020, which discloses that the binding affinity of IL-2 polypeptides with H16A and F42A substitutions for human IL-2Rα and IL-2Rβ was reduced by 110-fold and 3-fold, respectively, compared to wild-type IL2 binding, primarily due to faster off-rates for each of these interactions. TMPs containing such variants, including variants that exhibit reduced binding to IL-2Rα and IL-2Rβ, have demonstrated the ability to preferentially bind and activate IL-2 receptors on T cells that contain a target TCR specific for a peptide epitope on the TMP, and are therefore less likely to deliver IL-2 to non-target T cells, i.e., T cells that do not contain a TCR that specifically binds to a peptide epitope on the TMP. That is, binding of the IL-2 variant MOD to its costimulatory polypeptide on a T cell is substantially driven by binding of the MHC epitope portion rather than binding of IL-2. In some examples, the IL-2 variant MODs of the present disclosure exhibit reduced binding to IL-2Rγ.This reduction in binding to IL-2Rγ may be in addition to the reduction in binding to IL-2Rα and / or IL-2Rβ.

[0122] In some examples, a suitable variant IL-2 polypeptide has the following amino acid sequence: The variant IL-2 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025515473000008.tif17149. That is, the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2, but with H16A and F42A substitutions (shown in bold). Alternatively, the aforementioned sequences may be used with substitutions other than Ala at H16 and / or F42, e.g., H16T may be used in place of H16A. In some examples, the variant IL-2 polypeptide present in the TMP has the following amino acid sequence: In some examples, the variant IL-2 polypeptide present in the TMP comprises the following amino acid sequence: TIFF2025515473000010.tif23154. In some examples, the TMP comprises two copies of such a variant IL-2 polypeptide joined by a linker.

[0123] As described above, the TMP comprises an MHC class I heavy chain polypeptide. In some examples, the TMP comprises an MHC class I HLA-A allele heavy chain. In some examples, the MHC class I heavy chain polypeptide comprises the following HLA-A heavy chain amino acid sequence: It includes 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% amino acid sequence identity to TIFF2025515473000011.tif30159.

[0124] In some examples, the MHC class I heavy chain polypeptide comprises an Ala at position 84 and a Cys at position 236. Thus, for example, in some examples, the MHC class I heavy chain polypeptide comprises the following HLA-A heavy chain amino acid sequence: TIFF2025515473000012.tif30159 (amino acid 84 is Ala and amino acid 236 is Cys) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0125] As mentioned above, the TMP comprises an Ig Fc polypeptide. The Ig Fc polypeptide is also referred to herein as "Fc polypeptide". The Ig Fc polypeptide of the TMP can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc., or a variant of a wild-type Ig Fc polypeptide. The variant includes a natural variant, a non-natural variant, and a combination thereof. For example, the Ig Fc can be a variant of an Fc polypeptide, such as human IgG1 Fc, which has reduced or substantially eliminated ability to affect complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

[0126] In some examples, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide. For example, in some examples, the Fc polypeptide present in the TMP has the following human IgG1 Fc polypeptide amino acid sequence: Contains 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% amino acid sequence identity to TIFF2025515473000013.tif30157. Such IgG1 Fc polypeptides may or may not include a C-terminal lysine.

[0127] In some examples, the Fc polypeptide present in the TMP has the following human IgG1 Fc polypeptide amino acid sequence: It includes 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% amino acid sequence identity to TIFF2025515473000014.tif23156.

[0128] In some examples, the Ig Fc polypeptide present in the TMP 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% amino acid sequence identity to the amino acid sequence of the Ig Fc polypeptide shown in Figure 3D, where amino acid 14 is Ala and amino acid 15 is Ala. Thus, in some examples, the Ig Fc polypeptide present in the TMP comprises the following Ig Fc amino acid sequence: TIFF2025515473000015.tif23159 (amino acid 14 is Ala and amino acid 15 is Ala) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0129] In some examples, the Ig Fc polypeptide present in the TMP has the following Ig Fc amino acid sequence: TIFF2025515473000016.tif23159 (amino acid 14 is Ala and amino acid 15 is Ala) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0130] The TMP can include one or more independently selected peptide linkers, i.e., linkers that include two or more consecutive amino acid moieties, where the one or more linkers are between any two components of the TMP, such as between one or more of: (i) a peptide epitope and a β2M polypeptide, (ii) a first IL-2 polypeptide and a second IL-2 polypeptide, (iii) an IL-2 polypeptide and an MHC class I heavy chain polypeptide, and (iv) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide. The one or more linkers are independently selected. Suitable linkers include, for example, (GGGGS)n (SEQ ID NO:29) (n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)), AAAGG (SEQ ID NO:24), and the like.

[0131] In some examples, the TMP comprises a first polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 4A and a second polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 3A.

[0132] In some cases, TMP may be a) the amino acid sequence: a first polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025515473000017.tif10159; b) the amino acid sequence: (SEQ ID NO:15) and a second polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0133] In some cases, TMP may be a) the amino acid sequence: A first polypeptide having the sequence TIFF2025515473000018.tif10159; b) the amino acid sequence: (SEQ ID NO:15) and a second polypeptide having the following structure:

[0134] In some cases, TMP may be a) the amino acid sequence: A first polypeptide having the sequence TIFF2025515473000019.tif10159; b) the amino acid sequence: (SEQ ID NO:18) and a second polypeptide having the following structure:

[0135] In some cases, TMP is dimerized.For example, in some cases, one or more interchain disulfide bonds are formed spontaneously between two Ig Fc polypeptides (e.g., two IgG1 Fc polypeptides) in two TMPs, thereby forming homodimers.Therefore, in some cases, TMP is a protein that includes two heterodimers as described above.

[0136] In some instances, the TMP is a homodimer comprising two heterodimers, each heterodimer comprising: a) a first polypeptide, comprising, in order from N-terminus to C-terminus: i) the HPV16 E7 epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); ii) a peptide linker, and iii) a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 The first polypeptide comprising: b) a second polypeptide, i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence set forth in SEQ ID NO:5; ii) a class I MHC heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8, and iii) an IgG1 Fc polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; and the second polypeptide comprising Including, the first and second polypeptides of each heterodimer are covalently linked to each other via a disulfide bond between the Cys residue at amino acid 12 of the β2M polypeptide and the Cys residue at amino acid 236 of the class I MHC heavy chain polypeptide; The two heterodimers are linked to each other by one or more disulfide bonds which link the IgG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer.

[0137] In some instances, the TMP is a homodimer comprising two heterodimers, each heterodimer comprising: a) a first polypeptide, comprising, in order from N-terminus to C-terminus: i) the HPV16 E7 epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); ii) a linker comprising the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 13), and iii) a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 The first polypeptide comprising: b) a second polypeptide, i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence set forth in SEQ ID NO:5; ii) an MHC class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8, and iii) an IgG1 Fc polypeptide comprising an amino acid sequence having 98% identity with the amino acid sequence set forth in SEQ ID NO: 9 (without C-terminal Lys) or SEQ ID NO: 10 (without C-terminal Lys) or SEQ ID NO: 16 (with C-terminal Lys) or SEQ ID NO: 17 (with C-terminal Lys); and the second polypeptide comprising Including, the first and second polypeptides of each heterodimer are covalently linked to each other via a disulfide bond between the Cys residue at amino acid 12 of the β2M polypeptide and the Cys residue at amino acid 236 of the class I MHC heavy chain polypeptide; the two heterodimers are linked to one another by one or more disulfide bonds linking the gG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer; The second polypeptide is i) two IL-2 polypeptides, ii) one of two copies of an IL-2 polypeptide and an MHC class I heavy chain polypeptide; and iii) MHC class I heavy chain polypeptide and IgG1 Fc polypeptide and a peptide linker independently selected between one or more of:

[0138] In some instances, the TMP is CUE-101, which is a homodimer comprising two heterodimers, each heterodimer comprising the following: a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14; b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 (comprising an Ig Fc polypeptide without a C-terminal Lys) or SEQ ID NO: 18 (comprising an Ig Fc polypeptide with a C-terminal Lys); Including, the first and second polypeptides of each heterodimer are covalently linked to each other via a disulfide bond between the Cys residue at amino acid 12 of the β2M polypeptide and the Cys residue at amino acid 236 of the class I MHC heavy chain polypeptide; The two heterodimers are linked to each other by one or more disulfide bonds which link the IgG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer.

[0139] In some examples, the TMP is a single-chain polypeptide or a homodimer of two single-chain polypeptides that contain the same amino acid sequence. For example, the TMP may be a single-chain polypeptide that includes: (i) an HPV16 E7 epitope that contains the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); (ii) a β2-microglobulin (β2M) polypeptide; (iii) one or more copies of an IL-2 polypeptide that contains the amino acid sequence set forth in SEQ ID NO: 5; (iv) a class I MHC heavy chain polypeptide; (v) a variant human IgG1 Fc polypeptide; and (vi) one or more independently selected linkers that connect the components of the single-chain polypeptide. The single-chain polypeptide may further include one or more disulfide bonds.

[0140] For example, TMP is a homodimer comprising two single chain polypeptides, each single chain polypeptide comprising: (i) an HPV16 E7 epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4; (iii) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence set forth in SEQ ID NO:5; (iv) an MHC class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8; and (v) a variant IgG1 Fc polypeptide comprising amino acids having 98% identity to the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10; the two single chain polypeptides are linked to each other by one or more disulfide bonds linking the variant IgG1 Fc polypeptide of one single chain polypeptide to the variant IgG1 Fc polypeptide of the other single chain polypeptide; One or more independently selected linkers can connect the components of each single-chain polypeptide.

[0141] Method for generating mCTL The present disclosure provides a method for generating mCTLs. The method includes introducing one or more nucleic acids into a T cell, the one or more nucleic acids comprising (i) a polypeptide (e.g., a CAR) comprising an antigen-binding domain specific for a cancer-associated antigen; and (ii) an HPV E7 11-19 / 11-20 and a nucleotide sequence encoding a TCR. Methods for introducing such nucleic acids into T cells are known.

[0142] The present disclosure provides a method for the preparation of a cancer-associated antigen-binding polypeptide comprising: a) a first nucleic acid (which may be present in a recombinant expression vector) comprising a nucleotide sequence encoding a polypeptide (e.g., a CAR) comprising an antigen-binding domain specific for a cancer-associated antigen; and b) an HPV E7 11-19 / 20and a second nucleic acid (which may be present in a recombinant expression vector) that includes a nucleotide sequence encoding a TCR. The present disclosure provides a composition comprising: a) a first recombinant expression vector that includes a nucleotide sequence encoding a polypeptide (e.g., a CAR) that includes an antigen-binding domain specific for a cancer-associated antigen; and b) an HPV E7 11-19 / 20 and a second recombinant expression vector comprising a nucleotide sequence encoding a TCR. In some examples, the second nucleic acid or the second recombinant expression vector comprises a nucleotide sequence encoding the alpha and beta chain amino acid sequences shown in Figures 2A and 2B. In some examples, the second nucleic acid or the second recombinant expression vector comprises a nucleotide sequence encoding the alpha and beta chain amino acid sequences shown in Figures 2C and 2D.

[0143] The nucleic acid comprising the nucleotide sequence encoding CAR or TCR can be present in an expression vector, for example, a recombinant expression vector.Recombinant expression vectors and methods for making recombinant expression vectors are known.In some examples, for example, the recombinant expression vector is a viral construct, for example, a recombinant adeno-associated viral (AAV) construct, a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.In some examples, the nucleic acid comprising the nucleotide sequence encoding CAR or TCR is present in a recombinant lentiviral vector.In some examples, the nucleic acid comprising the nucleotide sequence encoding CAR or TCR is present in a recombinant AAV vector.

[0144] The nucleotide sequence encoding the CAR and / or the nucleotide sequence encoding the TCR can be operably linked to one or more transcriptional control elements, e.g., a promoter, e.g., a promoter that is functional in eukaryotic cells, which may be a constitutive promoter or an inducible promoter. Suitable promoters are known in the art, and any such promoter can be used.

[0145] In some examples, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a natural killer (NK) cell-specific promoter.For example, the CD4 gene promoter can be used (see, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7739, and Marodon et al. (2003) Blood 101: 3416).As another example, the CD8 gene promoter can be used.

[0146] The nucleic acid encoding the CAR or the nucleic acid encoding the TCR (e.g., a recombinant expression vector) can be introduced into the target T cell using any method that introduces a nucleic acid into a cell. Suitable methods include viral transfection (e.g., when the nucleic acid is a lentiviral vector or other viral vector that contains a nucleotide sequence encoding the CAR or TCR), electroporation, diethylaminoethyl (DEAE)-dextran mediated transfection, lipofection, and the like.

[0147] In some examples, one or more nucleic acids (e.g., recombinant expression vectors) are introduced into T cells in vitro to generate a mixed population of cells that includes mCTLs and non-modified T cells. In some examples, the mCTLs are separated from the non-modified T cells. In some examples, the mCTLs represent at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the total T cell population.

[0148] The method of separating mCTL from non-modified T cells is known in the art, and any known method can be used.For example, in some examples, the mixed population of cells including mCTL and non-modified T cells is contacted with immobilized HPV E7 peptide comprising amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), and mCTL is bound to immobilized HPV E7 peptide, thereby immobilizing mCTL, and immobilizing mCTL is separated from non-bound cells.In some examples, immobilized HPV E7 peptide is immobilized on a solid support such as beads.In some examples, immobilized HPV E7 peptide is bound to immobilized MHC class I polypeptide.

[0149] In some examples, the method includes contacting mCTL with TMP described herein in vitro. Such contacting step can increase the proliferation and / or activation of mCTL. In some examples, contacting mCTL with TMP increases the proliferation of mCTL by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the proliferation level in the absence of TMP (i.e., the proliferation level of mCTL not contacted with TMP). In some examples, after contact with TMP, the number of mCTLs increases by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more than 100-fold compared to the number of mCTLs not contacted with TMP.

[0150] Treatment method The present disclosure provides a method for treating cancer in an individual. The method generally involves administering a composition comprising mCTL of the present disclosure to an individual with cancer. In some examples, the method includes a) introducing a composition comprising mCTL and a composition comprising TMP as described herein to an individual with cancer. In some examples, the method includes a) introducing a composition comprising mCTL to an individual with cancer, and b) administering a TMP as described herein to the individual.

[0151] The disclosed methods include administering an effective amount of mCTLs that, when administered to an individual in need thereof in one or more doses, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or immune checkpoint inhibitors, as described below), reduces the overall tumor burden in the individual, i.e., the amount of cancer in the body, or alternatively keeps the total tumor burden in the patient relatively stable for a period of time sufficient for the patient to have confirmed "stable disease" as determined by standard RECIST criteria.

[0152] In some examples, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or an immune checkpoint inhibitor), reduces tumor size by a sufficient amount, and for a sufficient duration, for the patient to have a confirmed "partial response" as determined by standard RECIST criteria.

[0153] In some examples, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or an immune checkpoint inhibitor), reduces tumor size by a sufficient amount, and for a sufficient duration, for the patient to have a confirmed "complete response" as determined by standard RECIST criteria.

[0154] In some examples, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual (a tumor-bearing individual) in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or an immune checkpoint inhibitor), reduces tumor volume in the individual. For example, in some examples, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual (a tumor-bearing individual) in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or an immune checkpoint inhibitor), reduces tumor volume in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the tumor volume in the individual before or in the absence of administration of the mCTL. Tumor volume was determined using the formula (length x width x width) / 2, where length represents the maximum tumor diameter and width represents the perpendicular tumor diameter.

[0155] In some instances, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual in need thereof, extends the survival of the individual. For example, in some instances, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMP and / or an immune checkpoint inhibitor), extends the survival of the individual by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years, compared to the expected survival of the individual in the absence of administration of the mCTL.

[0156] In some examples, an effective amount of mCTL is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with a TMP and / or an immune checkpoint inhibitor), reduces circulating tumor DNA ("ctDNA") levels in a patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the ctDNA levels before or in the absence of mCTL administration. ctDNA levels can be determined using any known method (see, e.g., Cescon et al. (2020) Nature Cancer 1:276).

[0157] In some examples, the methods of the disclosure include a) contacting mCTLs with TMP in vitro and b) administering to an individual in need thereof an effective amount of a composition comprising mCTLs mixed with TMP. In some examples, the contacting step is performed for a period of time to allow expansion of mCTLs in vitro. For example, in some examples, the contacting step is performed for a period of 1 hour to 36 hours (e.g., 1 hour to 2 hours, 2 hours to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours, 12 hours to 18 hours, 18 hours to 24 hours, or 24 hours to 36 hours, or longer than 36 hours) prior to the administering step.

[0158] In some examples, the method of the present disclosure includes: a) administering an effective amount of mCTL to an individual in need thereof; and b) administering an effective amount of TMP to the individual. In some examples, the TMP is administered before administration of the mCTL. In some examples, the TMP is administered before and after administration of the mCTL. In some examples, the TMP is administered before and after administration of the mCTL.

[0159] In some examples, the TMP is administered after administration of the mCTLs, for example, about 1 hour to about 1 week after the administration step of the mCTLs. For example, in some examples, step (b) is performed 1 hour to about 2 hours, 2 hours to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours, 12 hours to 18 hours, 18 hours to 24 hours, 24 hours to 2 days, 2 days to 4 days, or 4 days to 1 week after step (a). The TMP can then be re-administered periodically as determined by the patient's physician to maintain a desired level of mCTLs in the patient. For example, the TMP may be administered weekly, every 2 weeks, every 3 weeks, monthly, or less frequently than monthly. The TMP may also be administered more frequently after the initial administration of the mCTLs, and then less frequently once a desired level of mCTL cells has been reached.

[0160] In some examples, the methods of the present disclosure can include administering to a patient between 10 cells per kg of body weight and 10 cells per kg of body weight. 9 For example, in some instances, the methods of the present disclosure include administering mCTLs in amounts ranging from 10 cells per kg of body weight to 10 cells per kg of body weight. 2 Cells, 10 per kg body weight 2 Cells ~ 10 per kg body weight 3 Cells, 10 per kg body weight 3 Cells ~ 10 per kg body weight 4 Cells, 10 per kg body weight 4 Cells ~ 10 per kg body weight 5 Cells, 10 per kg body weight 5 Cells ~ 10 per kg body weight 6 Cells, 10 per kg body weight 6 Cells ~ 10 per kg body weight 7 Cells, 10 per kg body weight 7 Cells ~ 10 per kg body weight 8 cells or 10 per kg of body weight 8 Cells ~ 10 per kg body weight 9 In some examples, a suitable number of mCTLs is 10 cells per kg of body weight, 10 cells per kg of body weight, or 10 cells per kg of body weight. 2 Cells, 10 per kg body weight 3 Cells, 10 per kg body weight 4Cells, 10 per kg body weight 5 Cells, 10 per kg body weight 6 Cells, 10 per kg body weight 7 Cells, 10 per kg body weight 8 cells, and 10 per kg of body weight 9 In some examples, the method comprises administering to the patient a cell number not greater than 10 per kg of body weight selected from the group consisting of: 7 In some embodiments, the method comprises administering a composition comprising an mCTL in an amount of 10 cells or less per kg of body weight. 6 In some embodiments, the method comprises administering a composition comprising an mCTL in an amount of 10 cells or less per kg of body weight. 5 This involves administering a composition comprising a subcellular amount of mCTL.

[0161] When the methods of the present disclosure include administering TMP, TMP may be administered in an amount of from 0.1 mg / kg to 20 mg / kg of body weight per dose, e.g., from 0.1 mg / kg to 10 mg / kg of body weight, e.g., from 0.5 mg / kg to 5 mg / kg of body weight, from 1 mg / kg to 5 mg / kg of body weight, from 5 mg / kg to 10 mg / kg of body weight, from 10 mg / kg to 15 mg / kg of body weight, from 15 mg / kg to 20 mg / kg of body weight, although doses beyond this exemplary range are contemplated, particularly in consideration of the aforementioned factors. For example, TMP can be administered in an amount of from about 1 mg / kg to 5 mg / kg of body weight, from about 5 mg / kg to about 10 mg / kg of body weight, from about 10 mg / kg to about 15 mg / kg of body weight, or from about 15 mg / kg to about 20 mg / kg of body weight. If the regimen is a continuous infusion, it may range from 1 μg to 10 mg per kilogram of body weight per minute.

[0162] When using the above-mentioned TMPs, e.g., homodimers comprising two heterodimers, each heterodimer comprising two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence set forth in SEQ ID NO:5, such homodimers may be administered in an amount of 4 mg / kg body weight or less, e.g., 3 mg / kg body weight, 2 mg / kg body weight, 1 mg / kg body weight, or less than 1 mg / kg body weight, as determined by the patient's physician in order to maintain a desired level of mCTL in the patient.

[0163] In general, when using the combination therapy of mCTL and TMP, mCTL can be administered at least one order of magnitude lower than the dose in the absence of administration of TMP, because TMP can then increase the number of mCTL in patient.In some cases, mCTL can be administered at least two orders of magnitude lower than the dose in the absence of administration of TMP.In some cases, mCTL can be administered at least three orders of magnitude lower than the dose in the absence of administration of TMP.

[0164] For example, according to the FDA-approved package insert, when CAR-T cells are genetically modified to produce Yescarta®, they bind to the CD19 + The number of CAR-T cells administered in a monotherapy regimen to an individual with cancer (e.g., B-cell lymphoma, such as follicular lymphoma, large B-cell lymphoma) is about 2×10 per kg of body weight. 6 CAR-positive viable T cells ~ up to 2 x 10 8 However, in combination with TMP, a smaller number, e.g., an order of magnitude less, e.g., about 2×10 per kg of body weight, is obtained. 5 CAR-positive viable T cells ~ up to 2 x 10 7 Approximately 2 x 10 CAR-positive viable T cells per kg of body weight 4 CAR-positive viable T cells ~ up to 2 x 10 6 viable CAR-positive T cells, or approximately 2 x 10 per kg of body weight 3 CAR-positive viable T cells ~ up to 2 x 10 5A patient may be administered Yescarta® CAR-T cells, a number of CAR-positive viable T cells. In this manner, the need for lymphodepletion prior to administration of Yescarta® CAR-T cells may be reduced or substantially eliminated. Similarly, the need for treatment with recombinantly produced IL-2, e.g., aldesleukin (Proleukin®), following administration of Yescarta® CAR-T cells may also be reduced or substantially eliminated. Initial administration of fewer Yescarta® CAR-T cells may also reduce one or more of the adverse side effects (e.g., cytokine release syndrome and / or neurotoxicity) associated with administration of such large numbers of Yescarta® CAR-T cells. Similar results and benefits may be achieved by administering TMP with fewer CAR-T cells, e.g., at least one order of magnitude, at least two orders of magnitude, or at least three orders of magnitude less, than the number indicated in the FDA-approved package insert for other CAR-T therapies, such as Tecartus®, Kymriah®, Abecma®, or Breyanzi®, as well as future approved CAR-T therapies.

[0165] In some cases, the disclosed method for treating cancer does not include lymphodepleting regimen. In other words, in some cases, the individual treated with mCTL does not undergo lymphodepleting chemotherapy. Lympho-depleting chemotherapy can include, for example, administration of cyclophosphamide and fludarabine before injecting T cells (for example, 2 to 7 days before injecting T cells).

[0166] In some examples, the disclosed methods for treating cancer do not include administering recombinantly produced IL-2, such as aldesleukin (Proleukin®), following administration of the mCTLs.

[0167] In some examples, the disclosed methods for treating cancer do not include administering (i) a lymphodepletion regimen, or (ii) recombinantly produced IL-2, such as aldesleukin (Proleukin®), following administration of the mCTLs.

[0168] A certain amount of mCTL can be administered to an individual in need thereof via any of a variety of administration routes.For example, the composition comprising mCTL can be administered via intramuscular, intravenous, peritumoral or intratumoral administration routes.Similarly, when TMP is administered to an individual, TMP can be administered to an individual via any of a variety of administration routes.For example, the composition comprising TMP (or dimerized TMP) can be administered via intramuscular, intravenous, peritumoral or intratumoral administration routes.

[0169] Combination therapy In some examples, the disclosed method for treating cancer in an individual includes a) administering mCTL to the individual, and b) administering at least one additional therapeutic agent or treatment to the individual. In some examples, the disclosed method for treating cancer in an individual includes a) administering mCTL and TMP to the individual, and b) administering at least one additional therapeutic agent or treatment to the individual. In some examples, the disclosed method for treating cancer in an individual includes a) administering mCTL and TMP to the individual, and b) administering TMP to the individual, and c) administering at least one additional therapeutic agent or treatment to the individual. Suitable additional therapeutic agents include, but are not limited to, small molecule cancer chemotherapeutic agents and immune checkpoint inhibitors. Suitable additional therapeutic agents include, for example, radiation, surgery (e.g., surgical removal of tumor), and the like.

[0170] The therapeutic methods of the present disclosure may include co-administration of a CART-MP and at least one additional therapeutic agent. By "co-administration" it is meant that both the CART-MP and the at least one additional therapeutic agent are administered to an individual, not necessarily at the same time, to achieve a therapeutic effect that is a result of administering both the CART-MP and the at least one additional therapeutic agent. The administration of the CART-MP and the at least one additional therapeutic agent may be substantially simultaneous, for example, the CART-MP may be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the at least one additional therapeutic agent. In some examples, the CART-MP is administered to an individual who is undergoing treatment with at least one additional therapeutic agent or has been treated with at least one additional therapeutic agent. The administration of the CART-MP may occur at different times and / or at different frequencies.

[0171] By way of example, a therapeutic method of the disclosure may include co-administration of a CART-MP and an immune checkpoint inhibitor, such as an antibody specific for an immune checkpoint. By "co-administration" it is meant that both a CART-MP and an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) are administered to an individual, not necessarily at the same time, to achieve a therapeutic effect that is the result of administering both a CART-MP and an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). Administration of the CART-MP and immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) may be substantially simultaneous, for example, the CART-MP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, within about 24 hours, within 1 week, 3 weeks, 3 weeks, 4 weeks, or within 1 month) after administration of the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). In some examples, a CART-MP of the disclosure is administered to an individual who is undergoing or has undergone treatment with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). Administration of the CART-MP and immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) may occur at different times and / or at different frequencies. If there is an established dosing interval for the checkpoint inhibitor, it may be possible to administer the CART-MP on the same day as the checkpoint inhibitor, depending on the interval. For example, in some instances, where the administration schedule of pembrolizumab is once every three weeks, the pharmaceutical composition comprising a CART-MP can be administered on the same day.

[0172] Exemplary immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some examples, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some examples, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0173] In some examples, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint polypeptide. In some examples, the anti-immune checkpoint antibody is a monoclonal antibody. In some examples, the anti-immune checkpoint antibody is humanized or deimmunized such that the antibody does not substantially elicit an immune response in humans. In some examples, the anti-immune checkpoint antibody is a humanized monoclonal antibody. In some examples, the anti-immune checkpoint antibody is a deimmunized monoclonal antibody. In some examples, the anti-immune checkpoint antibody is a fully human monoclonal antibody. In some examples, the anti-immune checkpoint antibody inhibits binding of an immune checkpoint polypeptide to a ligand of the immune checkpoint polypeptide. In some examples, the anti-immune checkpoint antibody inhibits binding of an immune checkpoint polypeptide to a receptor of the immune checkpoint polypeptide.

[0174] Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol-Myers Squibb), MEDI-6469 (MedImmune / AZ), CP-870,893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof. For example, in some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody. Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224.In some examples, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001. Suitable anti-PD1 antibodies are described in US Patent Publication No. 2017 / 0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some examples, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some examples, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, for example, Ribas et al. (2013) J. Clin. Oncol. 31:616-22. In some examples, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some examples, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some examples, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, for example, International Publication No. WO 2011 / 066389. For atezolizumab, see, for example, U.S. Patent No. 8,217,149. In some examples, the immune checkpoint inhibitor is an anti-TIGIT antibody that binds to T cell immune receptor with immunoglobulin and ITIM domains (TIGIT). In some examples, the anti-TIGIT antibody is BMS-986207 (Bristol-Myers Squibb). In some examples, the anti-TIGIT antibody is tiragolumab. In some examples, the anti-TIGIT antibody is EOS88448 (EOS-448). For example, see U.S. Patent Nos. 11,008,390 and 10,189,902, U.S. Patent Application Publication No. 2017 / 0088613, and International Publication No. 2019 / 137541.

[0175] Among such checkpoint inhibitors, antibodies against PD-1, PD-L1, TIGIT, LAG3 and CTLA-4 are the most common, with at least nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tisleizumab, and durvalumab approved by the FDA and / or regulatory agencies outside the U.S. An amount of the mCTLs of the present disclosure may also be co-administered with a combination of checkpoint inhibitors, such as a combination of (i) an antibody against PD-1 or PD-L1, and (ii) an antibody against CTLA-4.

[0176] Suitable subjects for treatment Suitable subjects for treatment using the methods of the present disclosure include individuals with cancer, including those who have been diagnosed with cancer, those who have been treated for cancer but have not responded to the treatment, and those who have been treated for cancer and initially responded but then become unresponsive to the treatment. Suitable subjects for treatment include individuals with cancer whose cancer cells express or overexpress a cancer associated antigen.

[0177] In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor, but whose disease has progressed despite such treatment. In some cases, the subject is an individual undergoing treatment with a cancer chemotherapeutic agent, or has been treated with a cancer chemotherapeutic agent. In some cases, the subject is an individual preparing to undergo treatment with an immune checkpoint inhibitor, undergoing treatment with an immune checkpoint inhibitor, or has been treated with an immune checkpoint inhibitor. In some cases, the subject is an individual preparing to undergo, undergoing, or has been treated with a cancer chemotherapeutic agent, radiation therapy, surgery, and / or another therapeutic agent.

[0178] Examples of Non-Limiting Aspects of the Disclosure Aspects including embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the disclosure are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0179] Behavior Set 1 Aspect 1. an amount of modified cytotoxic T cells ("mCTLs"), a) a T cell receptor (TCR) specific for an MHC class I polypeptide presenting a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) ("HPV E7"); 11-19 / 20 one or more nucleic acids comprising a nucleotide sequence encoding a TCR; b) one or more nucleic acids comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR); Including, the CAR comprises an antigen-binding domain specific for a cancer-associated antigen, and the percentage of targeted mCTL cells in the composition is greater than at least 1% of the total number of T cells in the composition; The amount of mCTLs.

[0180] Aspect 2. an amount of modified cytotoxic T cells ("mCTLs"), a) HPV E7 11-19 / 20 TCR and b) Chimeric antigen receptor (CAR) and Including, the CAR comprises an antigen-binding domain specific for a cancer-associated antigen, and the percentage of targeted mCTL cells in the composition is greater than at least 1% of the total number of T cells in the composition; The amount of mCTLs.

[0181] Aspect 3. an amount of modified cytotoxic T cells ("mCTLs"), a) HPV E7 11-19 / 20 A xenogeneic T cell receptor, TCR; b) Chimeric antigen receptor (CAR) and Including, the CAR comprises an antigen-binding domain specific for a cancer-associated antigen; The amount of mCTLs.

[0182] Aspect 4. The CAR, a) an extracellular domain comprising an antigen-binding domain; b) a transmembrane domain, and c) a cytoplasmic domain that contains an intracellular signaling domain An amount of mCTL according to any one of embodiments 1 to 3, comprising:

[0183] Aspect 5. The amount of the mCTL of embodiment 4, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

[0184] Aspect 6. The amount of the mCTL of embodiment 5, wherein the costimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

[0185] Aspect 7. 7. The composition according to any one of aspects 4 to 6, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0186] Aspect 8. The amount of the mCTL according to any one of aspects 1 to 7, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0187] Aspect 9. The amount of the mCTL according to any one of aspects 1 to 8, wherein the CAR is a single polypeptide chain CAR.

[0188] Aspect 10. The amount of the mCTL according to any one of embodiments 1 to 8, wherein the CAR comprises at least two polypeptide chains.

[0189] Aspect 11. 11. The amount of mCTL of any one of aspects 1-10, wherein the cancer associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, Glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0190] Aspect 12. The TCR (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG. 2A; (ii) having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in FIG. 2B; 12. An amount of mCTL according to any one of embodiments 1 to 11, comprising:

[0191] Aspect 13. The TCR (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG. 2C; (ii) having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in FIG. 2D; 12. An amount of mCTL according to any one of embodiments 1 to 11, comprising:

[0192] Aspect 14. The amount of mCTL according to any one of aspects 1 to 13, wherein the mCTL is a CD8+ T cell.

[0193] Aspect 15. The amount of mCTL according to any one of aspects 1 to 13, wherein the mCTL is a killer T cell or a killer innate-like T cell.

[0194] Aspect 16. 1. A composition comprising an amount of T cells, the amount of T cells comprises the mCTL according to any one of aspects 1 to 15; the percentage of said T cells that are mCTLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%; Optionally, a pharmaceutical composition suitable for administration to a human subject. The composition.

[0195] Aspect 17. below: (i) an amount of mCTL according to any one of aspects 1 to 15, or a composition according to aspect 16; (ii) a quantity of homodimers containing two heterodimers; A mixture comprising: Each heterodimer is a) a first polypeptide, i) a human papillomavirus epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), and ii) a first major histocompatibility complex (MHC) polypeptide, said first major histocompatibility complex polypeptide being a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence shown in FIG. 4B. The first polypeptide comprising: b) a second polypeptide, i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence shown in Figure 3B; iii) a second MHC polypeptide, which is an MHC class I heavy chain polypeptide comprising the amino acid sequence shown in FIG. 3C; and iv) Immunoglobulin (Ig) Fc Polypeptides and the second polypeptide comprising Including, the first polypeptide and the second polypeptide are covalently bound to each other via a disulfide bond; The mixture.

[0196] Aspect 18. the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence shown in FIG. The percent sequence identity can be determined by sequence alignment performed using BLAST, the Ig Fc polypeptide comprises Leu at position 14 and Leu at position 15, based on the amino acid numbering shown in FIG. said first and second polypeptides are covalently linked to each other via a disulfide bond between a Cys residue at amino acid 12 of said β2M polypeptide and a Cys residue at amino acid 236 of said class I MHC heavy chain polypeptide; the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide; The second polypeptide is a) a first copy of said IL-2 polypeptide, and a second copy of said IL-2 polypeptide; b) one of the two copies of said IL-2 polypeptide, and said MHC class I heavy chain polypeptide; and c) said MHC class I heavy chain polypeptide, and said Ig Fc polypeptide. and a peptide linker between one or more of Optionally, the homodimer comprises two heterodimers, each heterodimer comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. 18. The mixture according to embodiment 17.

[0197] Aspect 19. A method for producing an amount of mCTL according to any one of aspects 1 to 15, comprising: (i) a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific to a cancer-associated antigen; (ii) HPV E7 11-19 / 20 TCR and One or more nucleic acids encoding modifying a quantity of T cells by introducing The method.

[0198] Aspect 20. HPV E7 11-19 / 20 At least partially isolating target T cells that contain the TCR. Including, Optionally, HPV E7 11-19 / 20 the step of at least partially isolating target T cells comprising a TCR comprises binding said target T cells to an HPV16 E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2). 20. The method according to embodiment 19.

[0199] Aspect 21. The method of embodiment 20, wherein the HPV16 E7 peptide is immobilized on an insoluble support.

[0200] Aspect 22. The method of embodiment 21, wherein the insoluble support is a bead.

[0201] Aspect 23. The method of embodiment 19, wherein the HPV16 peptide is a peptide-bearing MHC class I multimer.

[0202] Aspect 24. The CAR, a) an extracellular domain comprising an antigen-binding domain; b) a transmembrane domain, and c) a cytoplasmic domain that contains an intracellular signaling domain 24. The method according to any one of aspects 19 to 23, comprising:

[0203] Aspect 25. 25. The method of embodiment 24, wherein the intracellular signaling domain comprises the signaling domain from the zeta chain of human CD3.

[0204] Aspect 26. 26. The method of embodiment 24 or 25, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

[0205] Aspect 27. 27. The method of embodiment 26, wherein the costimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

[0206] Aspect 28. 28. The method according to any one of aspects 19 to 27, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0207] Aspect 29. 29. The method according to any one of aspects 19 to 28, wherein the CAR is a single polypeptide chain CAR.

[0208] Aspect 30. 29. The method according to any one of embodiments 19 to 28, wherein the CAR comprises two polypeptide chains.

[0209] Aspect 31. 31. The method of any one of aspects 19 to 30, wherein the cancer associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, Glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0210] Aspect 32. 32. The method of any one of aspects 19-31, wherein said percentage of the total number of T cells in said composition that are target mCTLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.

[0211] Aspect 33. Prior to step (ii), a composition comprising an amount of T cells is subjected to in vitro or in vivo treatment with one of the following: a) a first polypeptide comprising: (i) an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a linker; and (iii) a β2 microglobulin polypeptide; b) a second polypeptide comprising (i) two copies of an IL-2 polypeptide, (ii) an MHC class I heavy chain polypeptide, and (iii) an immunoglobulin (Ig) Fc polypeptide; and A heterodimeric polypeptide comprising contacting the mixture with TMP containing Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, each heterodimeric polypeptide comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. A method according to any one of aspects 19 to 32.

[0212] Aspect 34. 34. The method according to any one of aspects 19 to 33, wherein at least 50% of the target T cells are CD8+ T cells.

[0213] Aspect 35. 35. The method according to any one of aspects 19 to 34, comprising enriching said T cells for CD8+ T cells between steps (i) and (ii).

[0214] Aspect 36. 36. The method according to any one of aspects 19 to 35, comprising enriching said T cells for CD8+ T cells between steps (ii) and (iii).

[0215] Aspect 37. 1. A method of treating cancer in an individual, comprising: introducing into said individual a composition comprising an amount of modified cytotoxic T cells according to any one of aspects 1 to 15, or a pharmaceutical composition prepared according to a method according to any one of aspects 16 to 33. The method comprising:

[0216] Aspect 38. The individual is provided with: a) a heterodimeric polypeptide, (i) a first polypeptide comprising an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), (ii) a linker, and (iii) a β2 microglobulin polypeptide; (i) two copies of an IL-2 polypeptide, (ii) an MHC class I heavy chain polypeptide, and (iii) a variant immunoglobulin (Ig) Fc polypeptide; and Including, Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, each heterodimeric polypeptide comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. the heterodimeric polypeptide, or b) a single-chain polypeptide comprising: (i) an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a β2 microglobulin polypeptide; (iii) one or more copies of a variant IL-2 polypeptide; (iv) an MHC class I heavy chain polypeptide; and (v) a variant immunoglobulin (Ig) Fc polypeptide. TMP containing either 38. The method of embodiment 37, further comprising administering a composition comprising:

[0217] Aspect 39. 39. The method of embodiment 37 or embodiment 38, wherein the step of administering a composition comprising an amount of genetically modified cytotoxic T cells comprises administering an amount of genetically modified cytotoxic T cells that is no more than a number selected from the group consisting of 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, 10 cells / kg body weight, and 10 cells / kg body weight.

[0218] Aspect 40. 40. The method of any one of aspects 37 to 39, wherein the step of administering a composition comprising an amount of genetically modified cytotoxic T cells comprises administering the amount of genetically modified cytotoxic T cells in an amount of 107 cells / kg or less of the body weight.

[0219] Aspect 41. The method according to any of aspects 37 to 40, wherein the individual has not undergone a lymphodepletion regimen prior to the introducing step.

[0220] Aspect 42. 42. The method of any one of aspects 37-41, wherein said administration is intramuscular, intravenous, peritumoral, or intratumoral.

[0221] Aspect 43. The method of any one of embodiments 37-42, further comprising administering to the individual one or more checkpoint inhibitors.

[0222] Aspect 44. 44. The method of embodiment 43, wherein the checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

[0223] Aspect 45. The method of embodiment 43, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1, or CTLA4.

[0224] Aspect 46. 46. ​​The method of embodiment 45, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, allelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, valilumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, KN035, and combinations thereof.

[0225] Aspect 47. The modified cells are 11-19 / 20 16. An amount of a modified cell according to any one of embodiments 1 to 15, comprising, instead of the TCR, a T cell receptor (TCR) specific for an MHC class I polypeptide that presents a peptide other than a human papillomavirus (HPV) E7 peptide comprising the amino acid sequence YMLDLQPETT or YMLDLQPET.

[0226] Aspect 48. 50. The amount of modified cells of any one of embodiments 1-15 or 47, wherein the modified cells comprise, instead of a CAR, an antigen binding domain other than a CAR.

[0227] Aspect 48. 50. The amount of modified cells of any one of aspects 1-15, 47, or 48, wherein the modified cells are cells other than T cells, e.g., NK cells, instead of T cells.

[0228] Behavior Set 2 Aspect 1. an amount of modified cytotoxic cells ("mCLs"), a) one or more nucleic acids comprising a nucleotide sequence encoding a given T cell receptor (TCR); b) an antigen-binding domain specific to a cancer-associated antigen; Including, the proportion of target mCL cells in the composition is at least 1% of the total number of T cells in said composition; The amount of mCL.

[0229] Aspect 2. an amount of modified cytotoxic cells ("mCLs"), a) HPV E7 11-19 / 20 TCR and b) an antigen-binding domain specific to a cancer-associated antigen; Including, the proportion of target mCL cells in the composition is greater than at least 1% of the total number of T cells in said composition; Optionally, the antigen-binding domain is a heterologous antigen-binding domain. The amount of mCL.

[0230] Aspect 3. an amount of modified cytotoxic cells ("mCLs"), a) HPV E7 11-19 / 20 A xenogeneic T cell receptor, TCR; b) a heterologous antigen-binding domain specific to a cancer-associated antigen; Including, The amount of mCL.

[0231] Aspect 4. the mCL comprises a CAR, The CAR, a) an extracellular domain comprising an antigen-binding domain; b) a transmembrane domain, and c) a cytoplasmic domain that contains an intracellular signaling domain An amount of mCL according to any one of embodiments 1 to 3, comprising:

[0232] Aspect 5. The amount of mCL of embodiment 4, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

[0233] Aspect 6. The amount of mCL according to embodiment 5, wherein said costimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

[0234] Aspect 7. 7. The composition according to any one of aspects 4 to 6, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0235] Aspect 8. The amount of mCL according to any one of aspects 1 to 7, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0236] Aspect 9. the mCL comprises a CAR, The CAR is a single polypeptide chain CAR. An amount of mCL according to any one of embodiments 1 to 8.

[0237] Aspect 10. the mCL comprises a CAR, The CAR comprises at least two polypeptide chains. An amount of mCL according to any one of embodiments 1 to 8.

[0238] Aspect 11. 11. The amount of mCL according to any one of aspects 1 to 10, wherein the cancer associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, Glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0239] Aspect 12. The TCR (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG. 2A; (ii) having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in FIG. 2B; An amount of mCL according to any one of embodiments 1 to 11, comprising:

[0240] Aspect 13. The TCR (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG. 2C; (ii) having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in FIG. 2D; An amount of mCL according to any one of embodiments 1 to 11, comprising:

[0241] Aspect 14. 14. The amount of mCL according to any one of aspects 1 to 13, wherein said mCL is a NK cell, a macrophage, or an ILC (innate lymphoid cell), which has a native, modified, or heterologous antigen-binding domain.

[0242] Aspect 15. 14. The amount of mCL according to any one of aspects 1 to 13, wherein said mCL is a T cell that is a classical T cell (alpha beta receptor expressing), a T cell expressing a gamma / delta TCR, a MAIT T cell of CD8 and CD4 phenotype, an MR-1 non-classical HLA-restricted, a killer T cell, or a killer innate-like T cell.

[0243] Aspect 16. 1. A composition comprising an amount of cells, The quantity of cells comprises mCL according to any one of aspects 1 to 15; the percentage of said cells that are mCLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%; Optionally, a pharmaceutical composition suitable for administration to a human subject. The composition.

[0244] Aspect 17. below: (i) an amount of mCL according to any one of aspects 1 to 15, or a composition according to aspect 16; (ii) a quantity of homodimers containing two heterodimers; A mixture comprising: Each heterodimer is a) a first polypeptide, i) a human papillomavirus epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), and ii) a first major histocompatibility complex (MHC) polypeptide, said first major histocompatibility complex polypeptide being a β2-microglobulin (β2M) polypeptide comprising the amino acid sequence shown in FIG. 4B. The first polypeptide comprising: b) a second polypeptide, i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence shown in Figure 3B; iii) a second MHC polypeptide, which is an MHC class I heavy chain polypeptide comprising the amino acid sequence shown in FIG. 3C; and iv) Immunoglobulin (Ig) Fc Polypeptides and the second polypeptide comprising Including, the first polypeptide and the second polypeptide are covalently bound to each other via a disulfide bond; The mixture.

[0245] Aspect 18. the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence shown in FIG. The percent sequence identity can be determined by sequence alignment performed using BLAST, the Ig Fc polypeptide comprises Leu at position 14 and Leu at position 15, based on the amino acid numbering shown in FIG. said first and second polypeptides are covalently linked to each other via a disulfide bond between a Cys residue at amino acid 12 of said β2M polypeptide and a Cys residue at amino acid 236 of said class I MHC heavy chain polypeptide; the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide; The second polypeptide is a) a first copy of said IL-2 polypeptide, and a second copy of said IL-2 polypeptide; b) one of the two copies of said IL-2 polypeptide, and said MHC class I heavy chain polypeptide; and c) said MHC class I heavy chain polypeptide, and said Ig Fc polypeptide. and a peptide linker between one or more of Optionally, the homodimer comprises two heterodimers, each heterodimer comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. 18. The mixture according to embodiment 17.

[0246] Aspect 19. A method for producing an amount of mCL according to any one of aspects 1 to 15, comprising: (i) an antigen-binding domain specific for a cancer-associated antigen; (ii) HPV E7 11-19 / 20 TCR and One or more nucleic acids encoding modifying a quantity of T cells by introducing The method.

[0247] Aspect 20. HPV E7 11-19 / 20 At least partially separating the target cells that contain the TCR. Including, Optionally, HPV E7 11-19 / 20 the step of at least partially isolating target cells comprising a TCR comprises binding said target cells to an HPV16 E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); 20. The method according to embodiment 19.

[0248] Aspect 21. The method of embodiment 20, wherein the HPV16 E7 peptide is immobilized on an insoluble support.

[0249] Aspect 22. 22. The method of embodiment 21, wherein the insoluble support is a bead.

[0250] Aspect 23. The method of embodiment 19, wherein the HPV16 peptide is a peptide-bearing MHC class I multimer.

[0251] Aspect 24. the mCL comprises a CAR, The CAR, a) an extracellular domain comprising an antigen-binding domain; b) a transmembrane domain, and c) a cytoplasmic domain that contains an intracellular signaling domain 24. The method according to any one of aspects 19 to 23, comprising:

[0252] Aspect 25. 25. The method of embodiment 24, wherein the intracellular signaling domain comprises the signaling domain from the zeta chain of human CD3.

[0253] Aspect 26. 26. The method of embodiment 24 or 25, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

[0254] Aspect 27. 27. The method of embodiment 26, wherein the costimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

[0255] Aspect 28. 28. The method according to any one of aspects 19 to 27, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0256] Aspect 29. the mCL comprises a CAR, The CAR is a single polypeptide chain CAR. A method according to any one of aspects 19 to 28.

[0257] Aspect 30. the mCL comprises a CAR, The CAR comprises two polypeptide chains. A method according to any one of aspects 19 to 28.

[0258] Aspect 31. 31. The method of any one of aspects 19 to 30, wherein the cancer associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, Glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0259] Aspect 32. 32. The method of any one of aspects 19 to 31, wherein the percentage of the total number of cells in the composition that are target mCLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.

[0260] Aspect 33. Prior to step (ii), a composition comprising a quantity of cells is subjected to, in vitro or in vivo, one of the following: a) a first polypeptide comprising: (i) an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a linker; and (iii) a β2 microglobulin polypeptide; b) a second polypeptide comprising (i) two copies of an IL-2 polypeptide, (ii) an MHC class I heavy chain polypeptide, and (iii) an immunoglobulin (Ig) Fc polypeptide; and A heterodimeric polypeptide comprising contacting the mixture with TMP containing Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, each heterodimeric polypeptide comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. A method according to any one of aspects 19 to 32.

[0261] Aspect 34. 34. The method according to any one of aspects 19 to 33, wherein at least 50% of the target cells are CD8+ T cells.

[0262] Aspect 35. 35. The method according to any one of aspects 19 to 34, comprising enriching the cells for CD8+ T cells between steps (i) and (ii).

[0263] Aspect 36. 36. The method according to any one of aspects 19 to 35, comprising enriching the cells for CD8+ T cells between steps (ii) and (iii).

[0264] Aspect 37. 1. A method of treating cancer in an individual, comprising: introducing into said individual a composition comprising an amount of modified cytotoxic cells according to any one of aspects 1 to 15, or a pharmaceutical composition prepared according to a method according to any one of aspects 16 to 33. The method comprising:

[0265] Aspect 38. The individual is provided with: a) a heterodimeric polypeptide, (i) a first polypeptide comprising an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), (ii) a linker, and (iii) a β2 microglobulin polypeptide; (i) two copies of an IL-2 polypeptide, (ii) an MHC class I heavy chain polypeptide, and (iii) a variant immunoglobulin (Ig) Fc polypeptide; and Including, Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, each heterodimeric polypeptide comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. the heterodimeric polypeptide, or b) a single-chain polypeptide comprising: (i) an HPV16 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO:1) or YMLDLQPET (SEQ ID NO:2); (ii) a β2 microglobulin polypeptide; (iii) one or more copies of a variant IL-2 polypeptide; (iv) an MHC class I heavy chain polypeptide; and (v) a variant immunoglobulin (Ig) Fc polypeptide. TMP containing either 38. The method of embodiment 37, further comprising administering a composition comprising:

[0266] Aspect 39. The step of administering a composition comprising an amount of genetically modified cytotoxic cells can be performed in a range of 10 cells per kg of body weight, 10 cells per kg of body weight, 10 2 Cells, 10 per kg body weight 3 Cells, 10 per kg body weight 4 Cells, 10 per kg body weight 5 Cells, 10 per kg body weight 6 Cells, 10 per kg body weight 7 Cells, 10 per kg body weight 8 Cells, 10 per kg body weight 9 The method of embodiment 37 or embodiment 38, comprising administering an amount of genetically modified cytotoxic cells up to a number selected from the group consisting of cells.

[0267] Aspect 40. The step of administering a composition comprising a quantity of genetically modified cytotoxic cells comprises administering a quantity of the composition comprising a quantity of genetically modified cytotoxic cells of at least 10 per kg of body weight. 7 40. The method according to any of embodiments 37-39, comprising administering a sub-cellular amount of the genetically modified cytotoxic cells.

[0268] Aspect 41. The method according to any of aspects 37 to 40, wherein the individual has not undergone a lymphodepletion regimen prior to the introducing step.

[0269] Aspect 42. 42. The method of any one of aspects 37-41, wherein said administration is intramuscular, intravenous, peritumoral, or intratumoral.

[0270] Aspect 43. The method of any one of embodiments 37-42, further comprising administering to the individual one or more checkpoint inhibitors.

[0271] Aspect 44. 44. The method of embodiment 43, wherein the checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

[0272] Aspect 45. The method of embodiment 43, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1, or CTLA4.

[0273] Aspect 46. 46. ​​The method of embodiment 45, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, allelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, valilumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, KN035, and combinations thereof. EXAMPLES

[0274] The following examples are presented to provide a more complete disclosure to those of skill in the art and are not intended to limit the scope of the invention as envisioned by the inventors, nor are they intended to represent that the following experiments are all or the only experiments performed.

[0275] Example 1 The CAR-T cell population was then treated with HPV E7 11-19 / 20 The mCTL composition is prepared by in vitro modification with the addition of one or more nucleic acids encoding a TCR. Alternatively, T cells are first transformed with HPV E7 11-19 / 20 The composition is modified by the addition of a nucleic acid encoding a TCR, followed by the addition of one or more nucleic acids encoding a CAR to prepare a composition comprising a population of mCTLs. The population of mCTLs is expanded in vitro prior to administration to a patient, for example using recombinantly produced IL-2 such as Proleukin® or a TMP such as CUE-101.

[0276] The patient does not undergo lymphodepletion therapy prior to administration of mCTL. The number of mCTL administered to the patient is at least an order of magnitude less than the dose expected in the absence of administration of TMP. After administration of mCTL, the patient is administered one or more doses of CUE-101 in an amount of up to about 4 mg per kg of body weight to increase the number of mCTL to a desired range and then to maintain the number of mCTL within the desired range. After administration of mCTL, the patient does not receive wild-type IL-2, such as Proleukin®. After administration of mCTL, the patient does not experience severe symptoms associated with cytokine release syndrome.

[0277] Example 2 Example 2 is carried out in the same manner as Example 1, except that prior to administration of mCTLs, the patient is administered a single dose of CUE-101 in an amount of up to about 4 mg per kg of body weight. After administration of mCTLs, the patient is administered one or more additional doses of CUE-101 in an amount of up to about 4 mg per kg of body weight to increase the number of mCTLs to a desired range and / or to maintain the number of mCTLs within a desired range. After administration of mCTLs, the patient does not receive wild-type IL-2, such as Proleukin®. After administration of mCTLs, the patient does not experience severe symptoms associated with cytokine release syndrome.

[0278] Example 3 The composition of mCTL is HPV E7 11-19 / 20 Yescarta containing nucleic acid encoding the TCR (登録商標) It is prepared to contain CAR-T cells.

[0279] The patient does not undergo lymphodepletion therapy prior to administration of the mCTLs. Optionally, prior to administration of the mCTLs to the patient, the patient is administered a single dose of CUE-101 in an amount of up to about 4 mg / kg of body weight.

[0280] The number of Yescarta® CAR-T cells administered to patients will be up to approximately 2 x 10 per kg of body weight. 5 CAR-positive viable T cells, up to 2 x 10 7 CAR-positive viable T cells, optionally up to approximately 2 x 10 per kg of body weight 4 CAR-positive viable T cells, up to 2 x 10 6 mCTLs are viable CAR-positive T cells. After administration of mCTLs, the patient is administered multiple additional doses of CUE-101 in an amount of up to about 4 mg per kg of body weight to increase the number of mCTLs to a desired range and then to maintain the number of mCTLs within the desired range. After administration of mCTLs, the patient does not receive wild-type IL-2, such as Proleukin®. After administration of mCTLs, the patient does not experience severe symptoms associated with cytokine release syndrome.

[0281] Although the present invention has been described with reference to specific embodiments in this disclosure, those skilled in the art will recognize that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications can be made to the particular conditions, materials, compositions of matter, processes, process step(s) to adapt to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. A certain amount of modified cytotoxic T cells ("mCTLs"), a) One or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific to an MHC class I polypeptide that presents the human papillomavirus (HPV) E7 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), b) One or more nucleic acids containing a nucleotide sequence encoding a chimeric antigen receptor (CAR) Includes, The CAR comprises an antigen-binding domain specific to cancer-associated antigens, and the proportion of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition. The aforementioned amount of mCTL.

2. A certain amount of modified cytotoxic T cells ("mCTLs"), a) A T cell receptor (TCR) specific to an MHC class I polypeptide that presents the human papillomavirus (HPV) E7 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), b) Chimeric antigen receptor (CAR) and Includes, The CAR comprises an antigen-binding domain specific to cancer-associated antigens, and the proportion of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition. The aforementioned amount of mCTL.

3. A certain amount of modified cytotoxic T cells ("mCTLs"), a) A heterologous T cell receptor (TCR) specific to an MHC class I polypeptide that presents the human papillomavirus (HPV) E7 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), b) Chimeric antigen receptor (CAR) and Includes, The CAR includes an antigen-binding domain specific to cancer-related antigens. The aforementioned amount of mCTL.

4. The aforementioned CAR, a) Extracellular domain including the antigen-binding domain, b) Transmembrane region, and c) Cytoplasmic domains containing intracellular signaling domains A quantity of mCTL according to any one of claims 1 to 3, including the above.

5. The amount of mCTL according to claim 4, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

6. The amount of mCTL according to claim 5, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

7. The amount of mCTL according to claim 4, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

8. The amount of mCTL according to any one of claims 1 to 3, wherein the antigen-binding domain is a single-stranded Fv polypeptide or a nanobody.

9. The amount of mCTL according to any one of claims 1 to 3, wherein the CAR is a single polypeptide chain CAR.

10. The CAR comprises at least two polypeptide chains in an amount of mCTL according to any one of claims 1 to 3.

11. The amount of mCTL according to any one of claims 1 to 3, wherein the cancer-related antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, Glypican-3, GPC3, HER-2, Kappa immunoglobulin, LeY, LMP1, Mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

12. The aforementioned TCR, (i) An α-chain having at least 90% amino acid sequence identity with the α-chain amino acid sequence shown in Figure 2A, (ii) A β-chain having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in Figure 2B A quantity of mCTL according to any one of claims 1 to 3, including the above.

13. The aforementioned TCR, (i) An α-chain having at least 90% amino acid sequence identity with the α-chain amino acid sequence shown in Figure 2C, (ii) A β-chain having at least 90% amino acid sequence identity with the β-chain amino acid sequence shown in Figure 2D A quantity of mCTL according to any one of claims 1 to 3, including the above.

14. The amount of mCTLs according to any one of claims 1 to 3, wherein the mCTLs are CD8+ T cells.

15. A composition containing a certain amount of T cells, The aforementioned amount of T cells comprises mCTLs according to any one of claims 1 to 3, The percentage of T cells that are mCTLs is selected from the groups consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%. The aforementioned composition.

16. The composition according to claim 15, which is a pharmaceutical composition suitable for administration to human subjects.

17. below: (i) A composition comprising a certain amount of mCTLs according to any one of claims 1 to 3, or a certain amount of T cells containing said mCTLs, (ii) A certain amount of homodimer containing two heterodimers A mixture containing, Each heterodimer, a) The first polypeptide, i) Human papillomavirus epitopes comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), and ii) The first major histocompatibility complex (MHC) polypeptide, which is a β2-microglobulin (β2M) polypeptide having the amino acid sequence shown in Figure 4B. The first polypeptide, including, b) A second polypeptide, i) Two copies of the IL-2 polypeptide, each containing the amino acid sequence shown in Figure 3B. ii) A second MHC polypeptide, which is an MHC class I heavy chain polypeptide containing the amino acid sequence shown in Figure 3C, and iii) Immunoglobulin (Ig) Fc polypeptide The second polypeptide, including Includes, The first polypeptide and the second polypeptide are covalently bonded to each other via a disulfide bond. The aforementioned mixture.

18. The IgFc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity with the amino acid sequence shown in Figure 3D. The aforementioned % sequence identity can be determined by sequence alignment performed using BLAST. The IgFc polypeptide includes Leu at position 14 and Leu at position 15, based on the amino acid numbering shown in Figure 3D. The first and second polypeptides are covalently bonded to each other via a disulfide bond between the Cys residue of amino acid 12 of the β2M polypeptide and the Cys residue of amino acid 236 of the class I MHC heavy chain polypeptide. The first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide. The second polypeptide is a) A first copy of the IL-2 polypeptide, and a second copy of the IL-2 polypeptide, b) One of the two copies of the IL-2 polypeptide, and the MHC class I heavy chain polypeptide, and c) The MHC class I heavy chain polypeptide and the Ig Fc polypeptide A peptide linker is included between one or more of them. The mixture according to claim 17.

19. A method for producing an amount of mCTL as described in any one of claims 1 to 3, the following: (i) A chimeric antigen receptor (CAR) containing an antigen-binding domain specific to cancer-related antigens, ()) (?) | １１－１９／２０ ()) One or more nucleic acids that encode This involves a process that modifies a certain amount of T cells by introducing [a specific method]. The aforementioned method.

20. HPV E7 １１－１９／２０ A step of at least partially isolating target T cells containing TCRs. Includes, Optionally, the HPV E7 １１－１９／２０ The step of at least partially isolating target T cells containing TCRs includes binding the target T cells to an HPV16 E7 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), The method according to claim 19.

21. The method according to claim 20, wherein the HPV16 E7 peptide is immobilized on an insoluble support.

22. The method according to claim 21, wherein the insoluble support is a bead.

23. The method according to claim 19, wherein the HPV16 E7 peptide is a peptide-supported MHC class I polymer.

24. The aforementioned CAR, a) Extracellular domain including antigen-binding domain, b) Transmembrane region, and c) Cytoplasmic domains containing intracellular signaling domains The method according to claim 19, including the method described in claim 19.

25. The method according to claim 24, wherein the intracellular signaling domain includes a signaling domain from the zeta chain of human CD3.

26. The method according to claim 24, wherein the cytoplasmic domain comprises one or more costimulatory polypeptides.

27. The method according to claim 26, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB, and OX-40.

28. The method according to claim 19, wherein the antigen-binding domain is a single-stranded Fv polypeptide or a nanobody.

29. The method according to claim 19, wherein the CAR is a single polypeptide chain CAR.

30. The method according to claim 19, wherein the CAR comprises two polypeptide chains.

31. The aforementioned cancer-related antigens are AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, The method according to claim 19, wherein the CAR is selected from GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2, and optionally the CAR comprises an scFv that binds to CD19 or BCMA.

32. The method according to claim 19, wherein the proportion of the total number of T cells in the composition that are target CTLs is selected from the group consisting 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.

33. Prior to step (ii), prepare a composition containing a certain amount of T cells in vitro or in vivo, as follows: a) A first polypeptide comprising (i) an HPV16 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), (ii) a linker, and (iii) a β2 microglobulin polypeptide, b) A second polypeptide comprising (i) two copies of IL-2 polypeptide, (ii) major histocompatibility complex class I (MHC class I) heavy chain polypeptide, and (iii) immunoglobulin (Ig) Fc polypeptide. T cell regulatory polypeptides (TMPs) The method according to claim 19, wherein the composition is brought into contact with the object.

34. The method according to claim 19, wherein at least 50% of the target T cells are CD8+ T cells.

35. The method according to claim 19, comprising enriching the T cells with respect to CD8+ T cells between steps (i) and (ii).

36. The method according to claim 19, comprising enriching the T cells with respect to CD8+ T cells between steps (ii) and (iii).

37. A method for treating cancer in an individual, A step of introducing into the organism a composition comprising an amount of modified cytotoxic T cells as described in any one of claims 1 to 3. The method, including the method described above.

38. To the aforementioned individual, the following: a) A first polypeptide comprising (i) an HPV16 peptide containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), (ii) a linker, and (iii) a β2 microglobulin polypeptide, b) A second polypeptide comprising (i) two copies of IL-2 polypeptide, (ii) major histocompatibility complex class I (MHC class I) heavy chain polypeptide, and (iii) immunoglobulin (Ig) Fc polypeptide. TMP The process further includes administering a composition containing the following: Optionally, the homodimer comprises two heterodimers, each heterodimer comprising a first polypeptide containing the amino acid sequence described in SEQ ID NO: 14 and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 15 or SEQ ID NO:

18. The method according to claim 37.

39. administering a composition comprising a certain amount of the genetically modified cytotoxic T cells, which is a number of cells selected from the group consisting of 10 cells per kg body weight, 10 ２ cells per kg body weight, 10 ３ cells per kg body weight, 10 ４ cells per kg body weight, 10 ５ cells per kg body weight, 10 ６ cells per kg body weight, 10 ７ cells per kg body weight, 10 ８ cells, and 10 ９ cells per kg body weight, the method according to claim 37, comprising administering a certain amount of the genetically modified cytotoxic T cells that is a number not exceeding that selected from the group consisting of the above cells.

40. The step of administering the composition containing a certain amount of genetically modified cytotoxic T cells is performed at a rate of 10 per kg of body weight. ７ The method according to claim 37, comprising administering a certain amount of genetically modified cytotoxic T cells less than one cell in size.

41. The method according to claim 37, wherein the individual does not undergo a lymph depletion regimen before the introduction step.

42. The method according to claim 37, wherein the administration is intramuscular, intravenous, peritumoral, or intratumoral.

43. The method according to claim 37, further comprising the step of administering one or more checkpoint inhibitors to the individual.

44. The method according to claim 43, wherein the checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

45. The method according to claim 43, wherein the checkpoint inhibitor is an antibody specific to PD-1, PD-L1, or CTLA4.

46. The method according to claim 45, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pizilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, allerumab, ipilimumab, tremelimumab, pizilizumab, IMP321, MGA271, BMS-986016, lirirumab, urerumab, PF-05082566, IPH2101, MEDI-6469, CP-870, 893, mogamulizumab, valirumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, KN035, and combinations thereof.

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