Modified cytotoxic t cells and methods of use thereof

Abstract

The present disclosure provides a modified cytotoxic T cell (mCTL) wherein the mCTL comprises: a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) having specificity for an MHC class I polypeptide presenting a human papilloma virus (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), wherein the CAR comprises an antigen binding domain specific for a cancer-associated antigen. The present disclosure provides a method of producing the mCTL. The present disclosure provides a method of treating cancer comprising administering the mCTL to an individual in need thereof.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 336,041, filed on April 28, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference of Electronically Submitted Materials

[0004] A sequence listing is provided with this application as a sequence listing XML "CUEB-150WO_SEQ_LIST", which was created on April 23, 2023 and is 136,686 bytes in size. The contents of the sequence listing XML are incorporated herein by reference in their entirety. Background Art

[0005] Cell therapy products contain T cells that have been modified 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 successfully used to treat cancer. For example, CAR-T cells targeting CD19, BCMA, CD30, CD22, or CD20 have shown significant activity in clinical studies. However, CAR-T therapy also has disadvantages. Due to the large number of CAR-T cells administered, patients usually require a chemotherapy regimen to deplete lymphocytes before the CAR-T cells are administered. In addition, in order to maintain therapeutic levels of CAR-T cells after administration, patients may also need to be treated with aldesleukin. Treatment may result in serious adverse side effects. In addition, some patients may not achieve their desired or optimal outcomes due to suboptimal expansion or persistence of CAR-T cells in the body. Summary of the invention

[0006] The present disclosure includes disclosure of modified cytotoxic T cells (mCTL), wherein the mCTL may comprise: 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 polypeptide comprising 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”) human papillomavirus (HPV) E7 peptide (collectively referred to as “HPV E7 11-19 / 20TCR"); and b) one or more nucleic acids comprising a nucleotide sequence encoding an antigen binding polypeptide (e.g., CAR or other polypeptides comprising an antigen binding domain specific for a cancer-associated antigen). The present disclosure also includes methods of producing mCTLs. The present disclosure also provides methods of treating cancer, comprising administering mCTLs to an individual in need thereof. The present disclosure also includes having a nucleotide sequence other than HPV E7 11-19 / 20 Disclosure of mCTLs expressing TCRs other than TCRs, and disclosure of modified cells other than T cells (e.g., NK cells) that are modified to simultaneously present TCRs and CARs or to present other cancer-associated antigen binding domains. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a schematic diagram of the generation and use of mCTLs according to the present disclosure.

[0009] Figure 2A-2D Amino acid sequences of exemplary TCR alpha and beta chains are provided (SEQ ID Nos: 19-22).

[0010] Figure 3A-3D Provided is the amino acid sequence of a T cell modulating polypeptide (TMP) polypeptide (SEQ ID NO: 15) ( Figure 3A ) and the amino acid sequences of the components of the polypeptide (SEQ ID No: 5, 8 and 10) ( Figure 3B-Figure 3D ).

[0011] Figure 4A-4B The amino acid sequence of the TM polypeptide is provided (SEQ ID NO: 14) ( Figure 4A ) and the amino acid sequence of β2M polypeptide (SEQ ID NO:4) ( Figure 4B ).

[0012] Figure 5 Sequence identifiers (SEQ ID Nos: 1-18) are provided for certain amino acid sequences shown in the specification and figures.

[0013] definition

[0014] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0015] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0016] A polynucleotide or polypeptide has a certain "sequence identity" percentage with another polynucleotide or polypeptide, meaning that when compared, when comparing the two sequences, the percentage of the bases or amino acids is the same and is located in the same relative position. Sequence identity can be determined in many different ways. In order to determine sequence identity, various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) can be used to align sequences, and the methods and computer programs can be obtained on the world wide web in the following websites: ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J.Mol.Biol.215:403-10.

[0017] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues with similar side chains. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; a group of amino acids with acidic side chains consists of glutamate and aspartic acid; and a 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.

[0018] "T cells" include all types of immune cells that express CD3, including T helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), T regulatory cells (Treg) and NK-T cells.

[0019] The terms "expression construct" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert.

[0020] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., antibody and antigen) and is expressed as the dissociation constant (K D ). Affinity can be at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 1,000 times or more of the affinity of the antibody to an unrelated amino acid sequence. For example, the affinity of the antibody to the target protein can be about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM) or about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. With respect to antibodies and / or antigen-binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein. Unless otherwise stated herein, when the word "about" is used in reference to a numerical value, it means a range of ±10% of the stated numerical value, for example, "about 10" means a value of 9 to 11.

[0021] As used herein, the term "binding" (e.g., referring to the binding of a T cell regulatory polypeptide ("TMP") to a polypeptide on a T cell (e.g., a T cell receptor); or referring to the 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 direct association between two molecules due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Non-covalent binding interactions are typically characterized by a dissociation constant (KD) of less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 MM, less than 10 -13 M, less than 10 -14 M or less than 10 -15 M. "Affinity" refers to the strength of non-covalent binding, with increased binding affinity correlating with lower KD. "Specific binding" generally refers to an affinity of at least about 10 -7 M or higher (e.g., 5x10 -7 M, 10 -8 M, 5x10 -8 M, 10 -9"Non-specific binding" generally refers to binding with an affinity of less than about 10 -7 M binding (e.g., affinity of 10 -6 M, 10 -5 M, 10 -4 "Specific binding" refers to the binding of a ligand to a portion of a receptor other than its designated binding site or receptor). However, in some cases, such as the binding between a TCR and a peptide / MHC complex, the range of "specific binding" can be 1 μM to 100 μM or 100 μM to 1 mM. As used herein, "covalent binding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0022] The terms "treatment", "treating", and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms and / or may be therapeutic in terms of partially or completely curing a disease and / or an adverse effect attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease or symptom in a mammal, and includes: (a) preventing a disease or symptom from occurring in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed with the disease or symptom; (b) inhibiting the disease or symptom, i.e., preventing its development; and / or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of persistent diseases is of particular interest in cases where the treatment stabilizes or alleviates the patient's undesirable clinical symptoms. Such treatment is ideally performed before the affected tissue completely loses function. The therapy of the present invention will ideally be administered during the symptomatic stage of the disease and in some cases after the symptomatic stage of the disease.

[0023] The terms "individual", "subject", "host" and "patient" are used interchangeably herein and 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., cattle, sheep, pigs, horses, goats, etc.), and the like.

[0024] As used herein, the term "about" used in conjunction with an amount indicates that the amount may vary by 10% of the stated amount. For example, "about 100" means an amount of 90-110. When about is used in the context of a range, "about" used when referring to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and "about" used when referring to the higher amount of the range means that the higher amount includes an amount that is 10% higher than the higher amount of the range. For example, about 100 to about 1000 means that the range extends from 90 to 1100.

[0025] Unless otherwise specified, the term "substantially" is intended to encompass both "completely" and "largely but not completely". For example, an Ig Fc that "substantially does not induce ADCC" means an Ig Fc that does not induce ADCC at all or does not induce ADCC to a large extent. Unless otherwise specified, the term "substantially" as used herein means "completely or largely but not completely". As another example, a "substantially homogeneous population" means a population that is completely homogeneous or largely but not completely homogeneous.

[0026] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments 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 (scAb), single-domain antibodies (dAb), single-domain heavy chain antibodies, single-domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising antigen-binding (also referred to herein as antigen-binding) portions of antibodies and non-antibody proteins. Antibodies can be detectably labeled, for example, with radioisotopes, enzymes that generate detectable products, fluorescent proteins, etc. Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (members of biotin-avidin specific binding pairs), etc. The term also encompasses 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 a group of identical cells, all of which are produced by a single cell through repeated cell replication. That is, the cell clone produces only a single antibody species. Although monoclonal antibodies can be produced using hybridoma production technology, they can also be produced using other production methods known to those skilled in the art (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or divalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of the following four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0027] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin comprising immunoglobulin portions of different origins, wherein at least one portion comprises an amino acid sequence of human origin. For example, a humanized antibody may comprise a portion of an immunoglobulin derived from a non-human source (such as a mouse) having the desired specificity, and a portion of an immunoglobulin sequence derived from a human source (e.g., a chimeric immunoglobulin), the portion being chemically linked together by conventional techniques (e.g., synthesis) or prepared as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding a protein portion of a chimeric antibody can be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains, the immunoglobulin chains comprising a complementary determining region (CDR) derived from an antibody of non-human origin and a framework region of a light chain and / or a heavy chain derived from a human source (e.g., a CDR-grafted antibody with or without a framework change). Chimeric or CDR-grafted single-chain antibodies are also encompassed within the term humanized immunoglobulin. See, e.g., U.S. Pat. No. 4,816,567; European Patent No. 0,125,023 B1; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694 B1; WO 86 / 01533; European Patent No. 0,194,276 B1; U.S. Pat. No. 5,225,539; European Patent No. 0,239,400 B1; and European Patent Application No. 0,519,596 A1. For single-chain antibodies, see also U.S. Pat. No. 4,946,778; U.S. Pat. No. 5,476,786; and Bird et al. (1988) Science 242:423.

[0028] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V HH ) and are known to those skilled in the art. The nanobodies are derived from heavy chain-only antibodies found in camelids (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). Immunoglobulins lacking light polypeptide chains are found in the "Camelids" family. "Camelids" include Old World camelids (Bactrian camels and dromedaries) and New World camelids (e.g., alpacas (Llama paccos), llamas (Llama glama), guanicoes (Llama guanicoe) and llamas (Llama vicugna)). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH Antibody.

[0029] "Antibody fragments" comprise 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. Papainization of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its ability to crystallize easily. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link antigen.

[0030] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region is composed of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly and non-covalently bound. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six CDRs together confer antigen binding specificity to the antibody. However, even a single variable domain (or only half of an Fv containing three CDRs that are specific to an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of a complete binding site.

[0031] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab' in which the cysteine ​​residues of the constant domains have free thiol groups is referred to herein as Fab'-SH. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0032] 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 sequences of their constant domains. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. 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), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further divided into different types, such as IgG2a and IgG2b.

[0033] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the V H Domain and V L In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994).

[0034] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are comprised of the same polypeptide chain (V H -V L ) is a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in a single chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0035] 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 heavy and light chain polypeptides. CDRs have been described in Kabat et al. (1977) J. Biol. Chem. 252: 6609; Kabat et al., US Pept. 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, where the definitions include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, the application of any definition to refer to the CDR of an antibody or a grafted antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. Table 1 below lists the amino acid residues encompassing the CDRs as defined by the various references cited above for comparison.

[0036] Table 1: CDR Definition

[0037] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> <![CDATA[MacCallum 3 ]]> <![CDATA[V H CDR-1]]> 31-35 26-32 30-35 <![CDATA[V H CDR-2]]> 50-65 53-55 47-58 <![CDATA[V H CDR-3]]> 95-102 96-101 93-101 <![CDATA[V L CDR-1]]> 24-34 26-32 30-36 <![CDATA[V L CDR-2]]> 50-56 50-52 46-55 <![CDATA[V L CDR-3]]> 89-97 91-96 89-96

[0038] 1 The residue numbering follows the nomenclature of Kabat et al., 1991, supra

[0039] 2 The residue numbering follows the nomenclature of Chothia et al., supra

[0040] 3 The residue numbering follows the nomenclature of MacCallum et al., supra

[0041] As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" refer to the first CDR, the second CDR and the third CDR in the light chain variable region, respectively. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" refer to the first CDR, the second CDR and the third CDR in the heavy chain variable region, respectively. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" refer to the first CDR, the second CDR and the third CDR in the variable region of either chain, respectively.

[0042] As used herein, when the term "framework" is used to refer to an antibody variable region, it is intended to refer to all amino acid residues outside the CDR region within the antibody variable region. The variable region framework is typically a discontinuous amino acid sequence between about 100-120 amino acids in length, but only those amino acids outside the CDR are intended to be referenced. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs.

[0043] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is only for the purpose of describing particular embodiments, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0044] Where a range of values ​​is provided, it is understood that unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range, separated by one-tenth of the unit of the lower limit, and any other stated or intermediate value within the stated range are encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limits within the stated ranges. Where a stated range includes one or two limits, ranges excluding one or both of these included limits are also encompassed within the disclosure.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can also be used to practice or test the present disclosure, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials related to the cited publications.

[0046] It must be noted that, as used herein and in the appended claims, the singular forms "a, an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "modified T cell" includes a plurality of such T cells and reference to a "T cell modulating polypeptide" or TMP includes 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 also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "only," "only," and the like in conjunction with the recitation of the claimed elements, or for the use of a "negative" limitation.

[0047] It should be understood that, for clarity, certain features of the present disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present disclosure are specifically covered by the present disclosure and disclosed herein, as if each combination is individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically covered by the present disclosure and disclosed herein, as if each such subcombination is individually and explicitly disclosed herein.

[0048] The publications discussed herein are provided only for their disclosures prior to the date of filing of the present application. Nothing herein is interpreted as admitting that the present disclosure is not eligible to precede such publications disclosed previously. In addition, the publication date provided may be different from the actual publication date which may need to be confirmed separately. DETAILED DESCRIPTION

[0049] The present disclosure provides modified cytotoxic T cells (mCTLs), wherein the mCTLs may comprise: a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide, wherein the MHC class I polypeptide presents a polypeptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) ("HPV E7 11-20 TCR") or YMLDLQPET(SEQ IDNO:2)("HPV E7 11-19 TCR”) human papillomavirus (HPV) E7 peptide (collectively referred to as “HPV E7 11-19 / 20 TCR"); and b) one or more nucleic acids comprising a nucleotide sequence encoding an antigen binding polypeptide (e.g., CAR or other polypeptides comprising an antigen binding domain specific for a cancer-associated antigen). The present disclosure also includes methods of producing mCTLs. The present disclosure also provides methods of treating cancer, comprising administering mCTLs to an individual in need thereof. The present disclosure also includes having a nucleotide sequence other than HPV E7 11-19 / 20 Disclosure of mCTLs expressing TCRs other than TCRs, and disclosure of modified cells other than T cells (e.g., NK cells) that are modified to simultaneously present TCRs and CARs or to present other cancer-associated antigen binding domains.

[0050] Modified cytotoxic T cells

[0051] The present disclosure provides modified cytotoxic T cells (mCTLs), wherein the mCTLs 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 (e.g., a CAR or other polypeptide comprising an antigen binding domain specific for a cancer associated antigen).

[0052] A T cell receptor (TCR) specific for presenting an MHC class I polypeptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) of a human papillomavirus (HPV) E7 peptide is referred to herein as "HPV E7" 11-19 / 20 TCR”.

[0053] The present disclosure provides a method for producing a HPV E7 11-19 / 20 The present disclosure provides a method for treating cancer, comprising administering to an individual in need thereof a mCTL comprising HPV E7 11-19 / 20 The present disclosure also provides mCTLs comprising HPVE7 11-19 / 20 mCTLs that contain a TCR and an antigen-binding polypeptide (e.g., a CAR or other polypeptide comprising an antigen-binding domain specific for a cancer-associated antigen).

[0054] Therefore, the present disclosure provides mCTL, wherein the mCTL comprises: a) comprising a polypeptide encoding PV E7 11-19 / 20 The invention relates to a method for treating a cancer-associated antigen in a humanized, non-human 247 cell line comprising a nucleic acid comprising a nucleotide sequence encoding a TCR; and b) one or more nucleic acids comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen.

[0055] Therefore, the present disclosure provides mCTL, wherein the mCTL comprises: a) HPV E7 11-19 / 2011-19 / 20 TCR; and b) CAR, wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen.

[0056] Although mCTLs are shown herein to have HPV E7 11-19 / 20TCR, i.e., a T cell receptor (TCR) with specificity for MHC class I polypeptides presenting human papillomavirus (HPV) E7 peptides comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), but it should be understood that mCTLs may have alternative TCRs specific for different peptides, in which case TMPs presenting peptides specific for alternative TCRs will be used. Similarly, mCTLs are shown to include CARs, but mCTLs may be other cell therapy products that include antigen binding domains for cancer-associated antigens, where the antigen binding domains are not CARs. The present disclosure explicitly contemplates mCTLs that include TCRs specific for different peptides and / or antigen binding domains other than CARs. Similarly, although the present disclosure is primarily directed to modified cytotoxic T cells (mCTLs), such as CAR-T cells, it should be understood that the disclosure herein can be used to modify other types of cytotoxic cells, such as NK cells, macrophages; ILCs (innate lymphocytes), which have natural or modified antigen binding domains. T cells can include classical T cells (expressing αβ receptors) or T cells expressing γ / δ TCRs; CD8 phenotype and CD4 phenotype; MAIT T cells restricted to MR-1 nonclassical HLA, killer T cells, and killer innate-like T cells.

[0057] In some cases, mCTL is in vitro. The in vitro composition of the present disclosure may include a T cell population that may contain T cells other than mCTL. Such cells are referred to as "unmodified T cells". Therefore, the in vitro composition of the present disclosure may be a heterogeneous population comprising mCTL and unmodified T cells. In some cases, 1% to 20% of the total number of T cells in the composition are mCTL. In some cases, 1% to 5%, 5% to 10%, 10% to 15% or 15% to 20% of the total number of T cells in the composition are mCTL. In some cases, 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 of the total number of T cells in the composition are mCTL. In some cases, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of the total number of T cells in the composition are mCTLs. Thus, in some cases, the T cell population in the composition is a substantially homogeneous population of mCTLs. As described above, "substantially homogeneous population" means a population that is completely homogeneous or largely but not completely homogeneous.

[0058] An exemplary use of an in vivo composition comprising mCTL is schematically shown in Figure 1 In vitro cell populations were modified to express CARs specific for target cancer-associated antigens and to express heterologous HPV E7 11-19 / 20 TCR. The in vitro composition comprising the target modified T cell is then administered to an individual in need thereof, e.g., an individual with a cancer that expresses the target 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 comprises an immunomodulatory polypeptide that provides an HPV E7 peptide that binds to the HPV E7 peptide present in the TMP. 11-19 / 20 Activation of T cells with TCR. Modified target T cells (e.g., mCTL) will target cancer cells expressing cancer-associated antigens presented by CAR. TMP will activate the modified target T cells by binding to the TCR present on the modified target T cells. The activation of the modified target can provide one or both of the following: (i) increase direct killing of cancer cells; and (ii) increase the secretion of cytokines that promote the anti-cancer activity of resident immune cells. TMP can increase the in vivo proliferation of mCTL, thereby reducing the number of mCTLs required for anti-cancer therapy, and also reduce or substantially eliminate the need for lymphocyte clearance chemotherapy before administering mCTL, and / or reduce or substantially eliminate the use of recombinantly produced IL-2 (e.g., aldesleukin) after administering CAR-T cells. )The need for treatment.

[0059] Chimeric Antigen Receptor

[0060] As described above, the modified T cells are modified to express CARs specific for cancer-associated antigens. CARs typically include: 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 cases, CAR includes: a) an extracellular domain comprising an antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising: i) one or more co-stimulatory polypeptides; and ii) an intracellular signaling domain. In some cases, CAR includes a hinge region between the extracellular antigen binding domain and the transmembrane domain. Therefore, in some cases, CAR includes: 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 cases, 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 co-stimulatory polypeptides; and ii) an intracellular signaling domain.

[0061] Exemplary CAR structures are known in the art (see, e.g., WO 2009 / 091826; US20130287748; WO 2015 / 142675; WO 2014 / 055657; WO 2015 / 090229; and U.S. Pat. No. 9,587,020).

[0062] In some cases, CAR is a single polypeptide chain. In some cases, CAR comprises two polypeptide chains. Generally, any CAR structure known to those skilled in the art can be used to modify T cells in order to prepare a composition as disclosed herein.

[0063] CARs specific for a variety of tumor antigens are known in the art; for example, 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; Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et 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) 23(15):3153-3161), 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 CAR (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CAR (Wang et al., Cancer Immunol Res (2014) 2(2):154-166), mesothelin (MSLN)-specific CAR (Moon et al., Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CAR (Van Seggelen et al., Mol Ther (2015) 23(10):1600-1610), CD19-specific CAR (Axicabtagene ciloleucel) (Yescarta. TM ) and Tisagenlecleucel (Kymriah TM ). See also, J Hematol and Oncol (2018) 11:22, reviewing clinical trials of tumor-specific CARs; Heyman and Yan (2019) Cancers 11:pii:E191; Baybutt et al. (2019) Clin. Pharmacol Ther. 105:71.

[0064] For example, CAR-T therapies include (Akirensai), which is a CAR comprising a scFv that binds CD19. As a further example, (brexucabtagene autoleucel) is a CAR containing a scFv that binds CD19; (tisagenleucleucel) is a CAR comprising a scFv that binds CD19; and (idecabtagene vicleucel) comprises a scFv that binds BCMA; and (lisocabtagene maraleucel) is a CAR comprising a scFv that binds CD19.

[0065] Antigen binding domain

[0066] As mentioned above, CAR includes an extracellular domain containing an antigen binding domain. The antigen binding domain present in CAR can be any antigen binding polypeptide, wherein a variety of antigen binding domains are known to the skill domain. In some cases, the antigen binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camel antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and "camelization" antibody variable domains are suitable. In some cases, the antigen binding domain is a nanobody.

[0067] In some cases, the antigen bound by the antigen binding domain of the CAR is selected from the group consisting of: MUC1 polypeptide, LMP2 polypeptide, epidermal growth factor receptor (EGFR) vIII polypeptide, HER-2 / neu polypeptide, melanoma antigen family A 3 (MAGE A3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate specific membrane antigen; PSMA) polypeptide, carcinoembryonic antigen (CEA) polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma translocation breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP ) polypeptide, melanoma inhibitor of apoptosis (ML-IAP) polypeptide, epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2ETS 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 homologous 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 P450 1B1 (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 antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen; also known as apical , sperm protein 17 polypeptide, lymphocyte-specific protein tyrosine kinase (LCK) polypeptide, high molecular weight melanoma-associated antigen (HMW-MAA), A-kinase anchoring 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, leguminous polypeptide (LGMN1; also known as asparaginyl endopeptidase), with Ig and EGF homology domain-2 (Tie-2;Also known as angiopoietin-1 receptor) tyrosine kinase 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 β (PDGFβ) polypeptide, MAD-CT-2 polypeptide or Fos-related antigen-1 (FOSL) polypeptide. In some cases, the antigen is a human papillomavirus (HPV) antigen. In some cases, the antigen is an alpha-fetoprotein (AFP) antigen. In some cases, the antigen is a Wilms tumor-1 (WT1) antigen. ;

[0068] The antigen binding polypeptide of 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, etc. Cancer-associated antigens also include, for example, 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, 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-1 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, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin.

[0069] In some cases, the cancer-associated antigens bound by the antigen-binding polypeptide of CAR are 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 ligands, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI and VEGFR2. In some cases, cancer-associated antigens are BCMA. In some cases, cancer-associated antigens are MUC1. In some instances, the cancer associated antigen is CD 19. In some instances, the cancer associated antigen is AFP.

[0070] The VH and VL amino acid sequences of various cancer-associated antigen-binding antibodies are known in the art, as are the light chain CDRs and heavy chain CDRs of such antibodies. See, for example, Ling et al. (2018) Frontiers Immunol. 9:469; WO 2005 / 012493; US 2019 / 0119375; US2013 / 0066055. The following are non-limiting examples of antibodies that bind to cancer-associated antigens.

[0071] 1) Anti-Her2

[0072] In some instances, the anti-Her2 antibody comprises: a) 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 the following amino acid sequence:

[0073] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25); and b) a heavy 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 following amino acid sequence:

[0074] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:26).

[0075] In some instances, 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, an anti-Her2 antibody may comprise: a) 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 the following amino acid sequence: DIQMTQSPSSLSASVG DRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 27); and b) 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 the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 28). In some cases, the anti-Her2 antibody comprises, from N-terminus to C-terminus: 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 the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 28); b) a linker; and c) 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 the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQK PGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 27). Suitable linkers are described elsewhere herein, and include, for example, (GGGGS)n (SEQ ID NO: 29), wherein n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0076] In some cases, the anti-Her2 antibody comprises the VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and the VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. H CDR and V L CDRs are as defined by Kabat (see, e.g., Table 1 above; and Kabat 1991). H CDR and V L The CDRs are as defined by Chothia (see, e.g., Table 1, supra; and Chothia 1987).

[0077] For example, an anti-Her2 antibody may comprise a VLCDR1 having the amino acid sequence of RASQDVNTAVA (SEQ ID NO: 30); a VL CDR2 having the amino acid sequence of SASFLY (SEQ ID NO: 31); a VL CDR3 having the amino acid sequence of QQHYTTPP (SEQ ID NO: 32); a VH CDR1 having the amino acid sequence of GFNIKDTY (SEQ ID NO: 33); a VH CDR2 having the amino acid sequence of IYPTNGYT (SEQ ID NO: 34); and a VH CDR3 having the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 35).

[0078] In some cases, the anti-Her2 antibody is a scFv antibody. For example, the anti-Her2 scFv may comprise 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 following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 36).

[0079] As another example, in some cases, the anti-Her2 antibody comprises: a) 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 the following amino acid sequence:

[0080] (SEQ ID NO: 37); and b) 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 the following amino acid sequence:

[0081] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:38).

[0082] In some instances, the anti-Her2 antibody comprises the VL present in the light chain amino acid sequence provided above; and the VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody may comprise: a) 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 the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGV AWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 39); and b) 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 the following amino acid sequence: EVQLVESGG GLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYC ARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO:40).

[0083] In some cases, the anti-Her2 antibody comprises the VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and the VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. H CDR and V L CDRs are as defined by Kabat (see, e.g., Table 1 above; and Kabat 1991). H CDR and V L The CDRs are as defined by Chothia (see, e.g., Table 1, supra; and Chothia 1987).

[0084] For example, an anti-HER2 antibody may comprise a VLCDR1 having the amino acid sequence of KASQDVSIGVA (SEQ ID NO: 41); a VL CDR2 having the amino acid sequence of SASYRY (SEQ ID NO: 42); a VL CDR3 having the amino acid sequence of QQYYIYPY (SEQ ID NO: 43); a VH CDR1 having the amino acid sequence of GFTFTDYTMD (SEQ ID NO: 44); a VH CDR2 having the amino acid sequence of ADVNPNSGGSIYNQRFKG (SEQ ID NO: 45); and a VH CDR3 having the amino acid sequence of ARNLGPSFYFDY (SEQ ID NO: 46).

[0085] In some cases, the anti-Her2 antibody is a scFv. For example, in some cases, the anti-Her2 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence:

[0086] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK(SEQ ID NO:36).

[0087] 2) Anti-CD19

[0088] Anti-CD19 antibodies are known in the art; and the VH and VL or VH CDR and VLCDR of any anti-CD19 antibody can be included in CAR. See, for example, WO 2005 / 012493.

[0089] In some cases, 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 cases, 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 instances, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence of KASQSVDYDGDSYLN (SEQ ID NO:47); a VL CDR2 comprising the amino acid sequence of DASNLVS (SEQ ID NO:48); a VL CDR3 comprising the amino acid sequence of QQSTEDPWT (SEQ ID NO:49); a VH CDR1 comprising the amino acid sequence of SYWMN (SEQ ID NO:50); a VH CDR2 comprising the amino acid sequence of QIWPGDGDTNYNGKFKG (SEQ ID NO:51); and a VH CDR3 comprising the amino acid sequence of RETTTVGRYYYAMDY (SEQ ID NO:52).

[0090] In some instances, the anti-CD 19 antibody is a scFv. For example, in some instances, the anti-CD 19 scFv 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 following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 53).

[0091] 3) Anti-mesothelin

[0092] Anti-mesothelin antibodies are known in the art; and the VH and VL or VH CDR and VLCDR of any anti-mesothelin antibody can be included in the CAR. See, for example, US2019 / 0000944; WO 2009 / 045957; WO 2014 / 031476; USPN 8,460,660; US2013 / 0066055; and WO 2009 / 068204.

[0093] In some instances, an anti-mesothelin antibody comprises: a) 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 the following amino acid sequence:

[0094] DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS(SEQID NO:54); and

[0095] b) a heavy 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 following amino acid sequence:

[0096] QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:55).

[0097] In some instances, an anti-mesothelin antibody comprises the VL present in the light chain amino acid sequence provided above; and the VH present in the heavy chain amino acid sequence provided above. For example, an anti-mesothelin antibody may comprise: a) 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 the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTI SGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO:56); and b) 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 the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGII DPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 57).

[0098] In some instances, the anti-mesothelin antibody comprises VLCDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. H CDR and V L CDRs are as defined by Kabat (see, e.g., Table 1 above; and Kabat 1991). H CDR and V L The CDRs are as defined by Chothia (see, e.g., Table 1, supra; and Chothia 1987).

[0099] For example, an anti-mesothelin antibody may comprise a VL CDR1 having the amino acid sequence of TGTSSDIGGYNSVS (SEQ ID NO: 58); a VL CDR2 having the amino acid sequence of LMIYGVNNRPS (SEQ ID NO: 59); a VL CDR3 having the amino acid sequence of SSYDIESATP (SEQ ID NO: 60); a VHCDR1 having the amino acid sequence of GYSFTSYWIG (SEQ ID NO: 61); a VH CDR2 having the amino acid sequence of WMGIIDPGDSRTRYSP (SEQ ID NO: 62); and a VH CDR3 having the amino acid sequence of GQLYGGTYMDG (SEQ ID NO: 63).

[0100] The anti-mesothelin antibody may be a scFv. As a non-limiting example, the anti-mesothelin scFv may comprise the following amino acid sequence:

[0101]

[0102] Wherein VH CDR1, CDR2 and CDR3 are underlined; and VL CDR1, CDR2 and CDR3 are bold and underlined.

[0103] As a non-limiting example, an anti-mesothelin scFv may comprise the following amino acid sequence:

[0104] Wherein VH CDR1, CDR2 and CDR3 are underlined; and VL CDR1, CDR2 and CDR3 are bold and underlined.

[0105] 4) Anti-BCMA

[0106] Anti-BCMA (B cell maturation antigen) antibodies are known in the art; and the VH and VL or VH CDR and VL CDR of any anti-BCMA antibody can be included in CAR. See, for example, WO 2014 / 089335; US2019 / 0153061; and WO2017 / 093942.

[0107] In some instances, the anti-BCMA antibody comprises: a) 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 the following amino acid sequence:

[0108] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSSKSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:66); and

[0109] b) a heavy 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 following amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:67).

[0110] In some cases, 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 may comprise: a) 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 with the following amino acid sequence:

[0111] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDSLNGWVFGGGTKLTVLG (SEQ ID NO:68); and b) 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 the following amino acid sequence:

[0112] EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSV (SEQ ID NO: 69).

[0113] In some instances, the anti-BCMA antibody comprises the VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and the VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. H CDR and V L CDRs are as defined by Kabat (see, e.g., Table 1 above; and Kabat 1991). H CDR and V L The CDRs are as defined by Chothia (see, e.g., Table 1, supra; and Chothia 1987).

[0114] For example, an anti-BCMA antibody may comprise a VLCDR1 having the amino acid sequence of SSNIGSNT (SEQ ID NO: 70); a VL CDR2 having the amino acid sequence of NYH; a VLCDR3 having the amino acid sequence of AAWDDSLNGWV (SEQ ID NO: 71); a VH CDR1 having the amino acid sequence of GFTFGDYA (SEQ ID NO: 72); a VH CDR2 having the amino acid sequence of SRSKAYGGTT (SEQ ID NO: 73); and a VHCDR3 having the amino acid sequence of ASSGYSSGWTPFDY (SEQ ID NO: 74).

[0115] The anti-BCMA antibody may be a scFv. As a non-limiting example, the anti-BCMA scFv may comprise the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 75).

[0116] As another example, an anti-BCMA scFv may comprise the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 76).

[0117] In some instances, an anti-BCMA scFv may comprise a VL CDR1 having the amino acid sequence of SASQDISNYLN (SEQ ID NO:77); a VL CDR2 having the amino acid sequence of YTSNLHS (SEQ ID NO:78); a VL CDR3 having the amino acid sequence of QQYRKLPWT (SEQ ID NO:79); a VH CDR1 having the amino acid sequence of NYWMH (SEQ ID NO:80); a VH CDR2 having the amino acid sequence of ATYRGHSDTYYNQKFKG (SEQ ID NO:81); and a VH CDR3 having the amino acid sequence of GAIYNGYDVLDN (SEQ ID NO:82).

[0118] In some instances, the anti-BCMA antibody comprises: a) 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 the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCS ASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO:83).

[0119] In some instances, the anti-BCMA antibody comprises: a) a heavy 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 following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVL DNWGQGTLVTVSS (SEQ ID NO:84).

[0120] In some instances, an anti-BCMA antibody (e.g., an antibody referred to in the literature as belantamab) comprises a light chain comprising the amino acid sequence: DIQMTQSPSS LSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQG TKLEIKR (SEQ ID NO: 83); and a heavy chain comprising the amino acid sequence: QV QLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGL EWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSE DTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 84).

[0121] 4).

[0122] In some cases, the anti-BCMA antibody has a cancer chemotherapeutic agent attached to the antibody. For example, in some cases, the anti-BCMA antibody is GSK2857916 (berantuzumab-mafodotin), in which monomethyl auristatin F (MMAF) is attached to the anti-BCMA antibody belantamab via a maleimidocaproyl linker.

[0123] 5) Anti-MUC1

[0124] In some cases, the antigen binding polypeptide present in CAR is a single-chain Fv specific for MUC1. See, e.g., Singh et al. (2007) Mol. Cancer Ther. 6:562; Thie et al. (2011) PLoS One 6:e15921; Imai et al. (2004) Leukemia 18:676; Posey et al. (2016) Immunity 44:1444; EP3130607; EP3164418; WO 2002 / 044217; and US2018 / 0112007. In some cases, the antigen binding polypeptide present in CAR is a scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:85). In some cases, TTP is a scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 86). In some cases, the antigen binding polypeptide present in the CAR is a scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 87). In some cases, TTP is a scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 88). In some cases, the antigen binding polypeptide present in the CAR is a scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 89).

[0125] For example, an anti-MUCl 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., US2018 / 0112007.

[0126] As another example, an anti-MUCl antibody may comprise a VHCDR1 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., US 2018 / 0112007.

[0127] As another example, an anti-MUC1 antibody may comprise a VHCDR1 having the amino acid sequence of DYAMN (SEQ ID NO: 102); a VH CDR2 having the amino acid sequence of VISTFSGNINFNQKFKG (SEQ ID NO: 103); a VH CDR3 having the amino acid sequence of SDYYGPYFDY (SEQ ID NO: 104); a VL CDR1 having the amino acid sequence of RSSQTIVHSNGNTYLE (SEQ ID NO: 105); a VL CDR2 having the amino acid sequence of KVSNRFS (SEQ ID NO: 106); and a VL CDR3 having the amino acid sequence of (FQGSHVPFT (SEQ ID NO: 107). See, e.g., US 2018 / 0112007.

[0128] As another example, an anti-MUC1 antibody may comprise a VHCDR1 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., US2018 / 0112007.

[0129] 6) Anti-MUC16

[0130] In some cases, the antigen binding polypeptide present in the CAR is specific for a MUC16 polypeptide present on a cancer cell. See, e.g., US2018 / 0118848; and US2018 / 0112008. In some cases, the MUC16-specific antigen binding polypeptide is a scFv. In some cases, the MUC16-specific antigen binding polypeptide is a nanobody.

[0131] 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 VLCDR1 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., US2018 / 0118848.

[0132] 7) Examples of antigen binding domains

[0133] In some cases, a suitable CAR comprises a scFv specific for CD 19. For example, in some cases, the anti-CD 19 scFv 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 following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDY DGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 53).

[0134] In some cases, a suitable CAR comprises a scFv specific for mesothelin. For example, in some cases, an anti-mesothelin scFv 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 following amino acid sequence: QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGRYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATISCRASQSVSSNFAWYQQRPGQAPRLLIYDASNRATGIPPRFSGSGSGTDFTLTISSLEPEDFAAYYCHQRSNWLYTFGQGTKVDIK (SEQ ID NO: 64).

[0135] In some instances, the anti-mesothelin scFv 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 following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMH WVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRRTVVTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCQASQDISNSLNWYQQKAGKAPKLLIYDASTLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQHDNLPLTFGQGTKVEIK (SEQ ID NO: 65).

[0136] In some cases, a suitable CAR comprises a scFv specific for B cell maturation antigen (BCMA). For example, in some cases, the anti-BCMA scFv 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 following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 75).

[0137] In some instances, the anti-BCMA scFv 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 following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWY QQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 76).

[0138] Hinge area

[0139] As described 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 connection region (also referred to herein as a "hinge" or "spacer"), which provides structural flexibility and spacing for the flanking polypeptide region, and may be composed of natural or synthetic polypeptides. The hinge region may include a complete hinge region derived from an antibody of a different class or subclass from the CH1 domain. The term "hinge region" may also include regions derived from CD8 and other receptors, which provide similar functions in providing flexibility and spacing for the flanking regions.

[0140] The hinge region may be about 4 amino acids to about 50 amino acids in length, e.g., 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.

[0141] As a non-limiting example, the immunoglobulin hinge region may include one of the following amino acid sequences: DKTHT (SEQ ID NO: 117), CPPC (SEQ ID NO: 118), CPEPK SCDTPPPCPR (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 may comprise an amino acid sequence derived from human CD8; for example, the hinge region may comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 128) or a variant thereof.

[0142] Transmembrane domain

[0143] Any transmembrane (TM) domain that provides for insertion of the polypeptide into the cell membrane of a eukaryotic cell (eg, mammalian) is suitable for use. The transmembrane region of CAR can be derived from (i.e., at least comprises the following transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), 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, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, I TGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, C D11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD 84. CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NT B-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR and PAG / Cbp. The transmembrane domain may be synthetic, in which case it may comprise primarily hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0144] As a non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 129) can be used. Other non-limiting examples of suitable TM sequences include: a) CD8β-derived TM: LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 130); b) CD4-derived TM: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 131); c) CD3ζ-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: ALPAALAVISFLLGLGLGVACVL A (SEQ ID NO: 135).

[0145] Intracellular domain – co-stimulatory peptide

[0146] The intracellular part (cytoplasmic domain) of CAR may include 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 co-stimulatory polypeptides include, for example: 1) a 4-1BB polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: KRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 136); 2) a CD28 polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 137); 3) an ICOS polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 138); 4) an OX40 polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 139); 5) a BTLA polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: CCLRRHQGKQNEL SDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCF RMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAP TEYASICVRS (SEQ ID NO:140); 6) a CD27 polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPA CSP (SEQ ID NO:141);7) a CD30 polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: RRACRK RIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 142); 8) a GITR polypeptide having at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity to the following amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 143). NO143 / ); and 9) an HVEM polypeptide having at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the following amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 144). The length of the co-stimulatory polypeptide may be about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa. ;

[0147] Intracellular domain – signaling polypeptide

[0148] The intracellular portion of CAR may include a signaling polypeptide. Suitable signaling polypeptides include, for example, intracellular signaling polypeptides containing an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acids. In some cases, the intracellular signaling domain of the subject CAR comprises 1, 2, 3, 4 or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated by 6 to 8 amino acids from each other, for example (YX1X2L / I)(X3) n (YX1X2L / I), wherein n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid. In some cases, the intracellular signaling domain of CAR comprises 3 ITAM motifs.

[0149] A suitable intracellular signaling domain may be a portion containing an ITAM motif that is derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain may be a domain containing an ITAM motif from any protein containing an ITAM motif. Therefore, a suitable intracellular signaling domain need not contain the complete sequence of the complete protein from which it is derived. Examples of suitable polypeptides containing an ITAM motif include, but are not limited to: DAP12; FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3Z (CD3ζ); and CD79A (antigen receptor complex-associated protein α chain).

[0150] TCR

[0151] As described above, the mCTLs of the present disclosure comprise (e.g., express on their surface) antibodies against HPV E7 11-19 / 20 The TCR has specificity, i.e., a TCR specific for the HPV E7 peptide YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) forms a complex with a class I MHC polypeptide (MHC class I heavy chain polypeptide and β2 microglobulin (β2M) polypeptide). The TCR is heterologous to the mCTL; i.e., the TCR is not naturally present in the mCTL.

[0152] In some cases, the mCTL comprises a heterologous TCR comprising: (i) an α chain comprising Figure 2A The amino acid sequence of D224 α chain shown in has at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity; and (ii) a β chain comprising Figure 2B The amino acid sequence of D224 α chain shown in has at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity. In some cases, the mCTL comprises a heterologous TCR comprising (i) an α chain having Figure 2A and (ii) a β chain having Figure 2B The amino acid sequence shown in .

[0153] In some cases, the mCTL comprises a heterologous TCR comprising: (i) an α chain comprising Figure 2C The amino acid sequence of D224 α chain shown in has at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity; and (ii) a β chain comprising Figure 2DThe amino acid sequence of D224 α chain shown in has at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity. In some cases, the mCTL comprises a heterologous TCR comprising (i) an α chain having Figure 2C and (ii) a β chain having Figure 2D The amino acid sequence shown in .

[0154] Compositions containing MCTL

[0155] The present disclosure provides a composition comprising mCTL of the present disclosure. The present disclosure provides a composition comprising T cells, wherein the percentage of 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%.

[0156] The composition of the present disclosure may include one or more of the following in addition to the mCTL of the present disclosure: salts, e.g., NaCl, MgCl2, KCl, MgSO4, etc.; buffers, e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizers; detergents, e.g., non-ionic detergents such as Tween-20, etc.; protease inhibitors; glycerol; etc. The composition comprising mCTL may include providing a liquid culture medium for cell maintenance.

[0157] The composition may include one or more pharmaceutically acceptable additives, wherein a variety of additives are known in the art and need not be discussed in detail herein. For example, see Sheskey et al., "Handbook of Pharmaceutical Excipients" (2020), 9th edition (or latest edition) and / or "Remington: The Science and Practice of Pharmacy", 23rd edition (2020), 23rd edition (or latest edition).

[0158] The pharmaceutical composition may comprise an amount of mCTL of the present disclosure and a pharmaceutically acceptable excipient. In some cases, the subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile. For example, in some cases, the subject pharmaceutical composition will be 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 the presence of such detectable pyrogens and / or other toxins is within acceptable limits set by applicable regulatory agencies (e.g., the U.S. Food and Drug Administration (USF&DA)).

[0159] The present disclosure provides a container comprising the mCTL of the present disclosure, such as a liquid composition. 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.

[0160] In some cases, the mCTL is present in a liquid composition. Thus, the present disclosure provides compositions (e.g., liquid compositions, including pharmaceutical compositions) comprising mCTL. In some cases, the compositions of the present disclosure comprise: a) mCTL; and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, 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 the presence level of such detectable pyrogens and / or other toxins is within acceptable limits set by applicable regulatory agencies (e.g., USF&DA). Thus, the present disclosure provides a composition comprising: a) mCTL; and b) saline (e.g., 0.9% NaCl), wherein the composition is sterile and free of detectable pyrogens and / or other toxins, or wherein the presence level of such detectable pyrogens and / or other toxins is within acceptable limits set by applicable regulatory agencies (e.g., USF&DA). In some cases, the composition further comprises a TMP as described herein.

[0161] The present disclosure provides a mixture of mCTL of the present disclosure and TMP, wherein the TMP comprises an HPV16 peptide comprising an amino acid sequence of YMLDLQPETT (SEQ ID NO: 1). The present disclosure provides a mixture of mCTL of the present disclosure and TMP, wherein the TMP comprises an HPV16 peptide comprising an amino acid sequence of YMLDLQPET (SEQ ID NO: 2).

[0162] T cell regulatory polypeptide (TMP)

[0163] T cell modulatory polypeptides (TMPs) suitable for use herein include single-chain polypeptides or heterodimeric polypeptides comprising: (i) a peptide, such as 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 immunomodulatory 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 scaffold polypeptide, such as an immunoglobulin (Ig) Fc polypeptide.

[0164] For example, the TMP may comprise a heterodimeric polypeptide comprising: a) 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 significantly reduced ability to achieve complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0165] The β2M polypeptide may comprise 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 following β2M amino acid sequence:

[0166] In some cases, 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 following β2M amino acid sequence: IQRTPKIQVY

[0167]

[0168] wherein amino acid 12 is Cys. In some cases, the β2M polypeptide comprises the amino acid sequence

[0169] In some cases, the IL-2 polypeptide is a variant of a naturally occurring co-stimulatory protein, and the variant exhibits reduced affinity to its corresponding (homologous) co-stimulatory protein (e.g., IL-2R) on T cells compared to the affinity of the naturally occurring IL-2 polypeptide to the corresponding co-stimulatory protein (IL-2R). In some cases, the IL-2 variant exhibits reduced binding to IL-2Rα, thereby minimizing or substantially reducing the activation of IL-2 variants to Treg. Or or in addition, in some cases, IL-2 variant MOD exhibits reduced binding to IL-2Rβ, so that IL-2 variant MOD exhibits overall reduced affinity to IL-2R. In some cases, IL-2 variant MOD exhibits two characteristics, that is, it exhibits reduced or substantially no binding to IL-2Rα, and also exhibits reduced binding to IL-2Rβ, so that IL-2 variant polypeptides exhibit overall reduced affinity to IL-2R. For example, IL-2 variants with substitutions at H16 and F42 show reduced binding to IL-2Rα and IL-2Rβ. See, Quayle et al., Clin Cancer Res; 26(8) April 15, 2020, which discloses that IL-2 polypeptides with H16A and F42A substitutions have 110-fold and 3-fold reduced binding affinity for human IL-2Rα and IL-2Rβ, respectively, compared to wild-type IL2 binding, primarily due to faster dissociation rates for each of these interactions. TMPs comprising such variants (including variants exhibiting reduced binding to IL-2Rα and IL-2Rβ) show the ability to preferentially bind to and activate IL-2 receptors on T cells containing target TCRs specific for peptide epitopes on TMPs, and are therefore less likely to deliver IL-2 to non-target T cells, i.e., T cells that do not contain TCRs that specifically bind to peptide epitopes on TMPs. In other words, the binding of IL-2 variant MOD to its co-stimulatory polypeptide on T cells is substantially driven by the binding of the MHC-epitope portion rather than by the binding of IL-2. In some cases, the IL-2 variant MOD of the present disclosure exhibits reduced binding to IL-2Rγ. Reduced binding to IL-2Rγ may be associated with reduced binding to IL-2Rα and / or IL-2Rβ.

[0170] In some cases, a suitable 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 the following amino acid sequence:

[0171] That is, the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but has H16A and F42A substitutions (shown in bold). Alternatively, the aforementioned sequence can be used, but with substitutions other than Ala at H16 and / or F42, for example, H16T can be used instead of H16A. In some cases, the variant IL-2 polypeptide present in TMP comprises the following amino acid sequence:

[0172] In some cases, the variant IL-2 polypeptide present in TMP comprises the following amino acid sequence:

[0173] In some cases, the TMP comprises two copies of such a variant IL-2 polypeptide connected by a linker.

[0174] As described above, the TMP comprises an MHC class I heavy chain polypeptide. In some instances, the TMP comprises an MHC class I HLA-A allele heavy chain. In some instances, the MHC class I heavy chain polypeptide 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 following HLA-A heavy chain amino acid sequence:

[0175]

[0176] In some instances, the MHC class I heavy chain polypeptide comprises Ala at position 84 and Cys at position 236. Thus, for example, in some instances, the MHC class I heavy chain polypeptide 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 following HLA-A heavy chain amino acid sequence:

[0177]

[0178] wherein amino acid 84 is Ala and amino acid 236 is Cys.

[0179] As described above, the TMP comprises an Ig Fc polypeptide. The Ig Fc polypeptide is also referred to herein as an "Fc polypeptide". The Ig Fc polypeptide of the TMP may be a variant of human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc., or a wild-type Ig Fc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, Ig Fc may be a variant of an Fc polypeptide (such as a human IgG1 Fc polypeptide) that has a reduced or substantially eliminated ability to achieve complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0180] In some cases, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. For example, in some cases, the 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 following human IgG1 Fc polypeptide amino acid sequence: NO:9). Such IgG1 Fc polypeptides may or may not contain a C-terminal lysine.

[0181] In some cases, the 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 following human IgG1 Fc polypeptide amino acid sequence:

[0182] In some cases, the Ig Fc polypeptide present in the TMP comprises Figure 3D , wherein amino acid 14 is Ala and amino acid 15 is Ala. Thus, in some cases, 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 Ig Fc polypeptide amino acid sequence shown in:

[0183] wherein amino acid 14 is Ala and amino acid 15 is Ala.

[0184] In some cases, 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 following Ig Fc amino acid sequence:

[0185] wherein amino acid 14 is Ala and amino acid 15 is Ala.

[0186] The TMP may include one or more independently selected peptide linkers, i.e., linkers comprising a continuous stretch of two or more amino acids, wherein the one or more linkers are between any two components of the TMP, for example, between one or more of the following: (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), wherein n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); AAAGG (SEQ ID NO: 24); and the like.

[0187] In some cases, TMP contains Figure 4A a first polypeptide having at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity with the amino acid sequence shown in ; and Figure 3A 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 shown in .

[0188] In some cases, the TMP comprises: a) a first polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: YMLDLQPETTGGGGSGGGGSGGGGSIQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSF YLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 14);and b) a second polypeptide having at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTK KTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEAAAGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:15).;

[0189] In some cases, the TMP comprises: a) a first polypeptide having the following amino acid sequence: YMLDLQPETTGGGGSGGGGSGGGGSIQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 14);and b) a second polypeptide having the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEAAAGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:15).;

[0190] In some cases, the TMP comprises: a) a first polypeptide having the following amino acid sequence: YMLDLQPETTGGGGSGGGGSGGGGSIQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 14);and b) a second polypeptide having the following amino acid sequence: (SEQ ID NO: 18). ;

[0191] In some cases, TMP dimerizes. For example, in some cases, two Ig Fc polypeptides (e.g., two IgG1 Fc polypeptides) are spontaneously linked by one or more interchain disulfide bonds to form a homodimer. Thus, in some cases, TMP is a protein comprising two heterodimers, as described above.

[0192] In some cases, TMP is a homodimer comprising two heterodimers, wherein each heterodimer comprises:

[0193] a) The first polypeptide comprises, from N-terminus to C-terminus:

[0194] i) an HPV16 E7 epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2);

[0195] ii) a peptide linker; and

[0196] iii) a β2 microglobulin (β2M) polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4; and

[0197] b) a second polypeptide comprising:

[0198] i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence shown in SEQ ID NO: 5;

[0199] ii) a class I MHC heavy chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8; and

[0200] iii) an IgG1 Fc polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10,

[0201] wherein the first polypeptide and the second polypeptide 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, and

[0202] The two heterodimers are linked to each other via one or more disulfide bonds, which connect the IgG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer.

[0203] In some cases, TMP is a homodimer comprising two heterodimers, wherein each heterodimer comprises:

[0204] a) The first polypeptide comprises, from N-terminus to C-terminus:

[0205] i) an HPV16 E7 epitope comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2);

[0206] ii) a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13); and

[0207] iii) a β2 microglobulin (β2M) polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4; and

[0208] b) a second polypeptide comprising:

[0209] i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence shown in SEQ ID NO: 5

[0210] ii) an MHC class I heavy chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8; and

[0211] iii) an IgG1 Fc polypeptide comprising amino acids having 98% identity to the amino acid sequence shown in SEQ ID NO: 9 (excluding the C-terminal Lys), or SEQ ID NO: 10 (excluding the C-terminal Lys), or SEQ ID NO: 16 (including the C-terminal Lys), or SEQ ID NO: 17 (including the C-terminal Lys).

[0212] wherein the first polypeptide and the second polypeptide 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, and

[0213] wherein the two heterodimers are linked to each other via one or more disulfide bonds, wherein the disulfide bonds link the gG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer,

[0214] wherein the second polypeptide comprises an independently selected peptide linker between one or more of:

[0215] i) two IL-2 polypeptides;

[0216] ii) one of the two copies of an IL-2 polypeptide and an MHC class I heavy chain polypeptide; and

[0217] iii) MHC class I heavy chain polypeptide and IgG1 Fc polypeptide.

[0218] In some cases, the TMP is CUE-101, which is a homodimer comprising two heterodimers, wherein each heterodimer comprises:

[0219] a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 14;

[0220] b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 15 (which includes an Ig Fc polypeptide not including a C-terminal Lys) or SEQ ID NO: 18 (which includes an Ig Fc polypeptide including a C-terminal Lys);

[0221] wherein the first polypeptide and the second polypeptide 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, and

[0222] The two heterodimers are linked to each other via one or more disulfide bonds, which connect the IgG1 Fc polypeptide of one heterodimer to the variant immunoglobulin Fc polypeptide of the other heterodimer.

[0223] In some cases, the TMP is a single-chain polypeptide or a homodimer of two single-chain polypeptides comprising the same amino acid sequence. For example, the TMP can be a 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, (iii) one or more copies of an IL-2 polypeptide comprising the amino acid sequence shown 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 for connecting the single-chain polypeptide components. The single-chain polypeptide may further comprise one or more disulfide bonds.

[0224] For example, TMP is a homodimer comprising two single-chain polypeptides, wherein each single-chain polypeptide comprises (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.

[0225] wherein the two single-chain polypeptides are linked to each other via one or more disulfide bonds, wherein the disulfide bonds link the variant IgG1 Fc polypeptide of one single-chain polypeptide to the variant IgG1 Fc polypeptide of the other single-chain polypeptide, and

[0226] One or more independently selected linkers may connect the components of each single polypeptide chain.

[0227] Method for preparing MCTL

[0228] The present disclosure provides a method for preparing mCTL. The method comprises introducing one or more nucleic acids into T cells, wherein the one or more nucleic acids comprise a polypeptide encoding (i) an antigen binding domain specific for a cancer-associated antigen (e.g., CAR) and (ii) HPV E7 11-19 / 11-20 Nucleotide sequences of TCRs. Methods for introducing such nucleic acids into T cells are known.

[0229] The present disclosure provides a composition comprising: a) a first nucleic acid (which may be present in a recombinant expression vector) comprising a nucleotide sequence encoding a polypeptide comprising an antigen binding domain specific to a cancer associated antigen (e.g., CAR); and b) a second nucleic acid (which may be present in a recombinant expression vector) comprising a nucleotide sequence encoding an HPV E7 11-19 / 20 The present disclosure provides a composition comprising: a) a first recombinant expression vector comprising a nucleotide sequence encoding a polypeptide (e.g., CAR) comprising an antigen binding domain specific to a cancer-associated antigen; and b) a second recombinant expression vector comprising a nucleotide sequence encoding an HPV E7 11-19 / 20 In some cases, the second nucleic acid or the second recombinant expression vector comprises a nucleotide sequence encoding a TCR. Figure 2A The α chain and Figure 2B In some cases, the second nucleic acid or the second recombinant expression vector comprises a nucleotide sequence encoding Figure 2C The α chain and Figure 2D The nucleotide sequence of the β chain amino acid sequence shown in .

[0230] The nucleic acid comprising the nucleotide sequence encoding CAR or TCR may be present in an expression vector, such as a recombinant expression vector. Recombinant expression vectors and methods for preparing recombinant expression vectors are known. In some cases, for example, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus (AAV) construct, a recombinant adenovirus construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc. In some cases, the nucleic acid comprising the nucleotide sequence encoding CAR or TCR is present in a recombinant lentiviral vector. In some cases, the nucleic acid comprising the nucleotide sequence encoding CAR or TCR is present in a recombinant AAV vector.

[0231] The nucleotide sequence encoding the CAR and / or the nucleotide sequence encoding the TCR can be operably connected to one or more transcription control elements, such as a promoter, such as a promoter functional in a eukaryotic cell, wherein the promoter can be a constitutive promoter or an inducible promoter. Suitable promoters are known in the art and any such promoter can be used.

[0232] In some cases, 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.

[0233] Any method for introducing nucleic acid into cells can be used to introduce nucleic acid (e.g., recombinant expression vector) encoding CAR or encoding TCR into target T cells. Suitable methods include viral transfection (e.g., where nucleic acid is a lentiviral vector or other viral vectors comprising a nucleotide sequence encoding CAR or TCR), electroporation, diethylaminoethyl (DEAE)-dextran-mediated transfection, lipofection, etc.

[0234] In some cases, once one or more nucleic acids (e.g., recombinant expression vectors) are introduced into T cells in vitro, a mixed cell population comprising mCTL and unmodified T cells is generated. In some cases, mCTL are separated from unmodified T cells. In some cases, mCTL comprises 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.

[0235] Methods for separating mCTL from unmodified T cells are known in the art, and any known method can be used. For example, in some cases, a mixed cell population comprising mCTL and unmodified T cells is contacted with an immobilized HPV E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2), allowing mCTL to bind to the immobilized HPV E7 peptide, thereby immobilizing the mCTL, and separating the immobilized mCTL from the unbound cells. In some cases, the immobilized HPV E7 peptide is immobilized on a solid support, such as beads. In some cases, the immobilized HPV E7 peptide is bound to an immobilized MHC class I polypeptide.

[0236] In some cases, the method includes contacting in vitro mCTL with a TMP as described herein. Such a contact step can increase the proliferation and / or activation of mCTL. In some cases, 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 times), at least 2.5 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times or more than 100 times compared to the proliferation level in the absence of TMP (i.e., the proliferation level of mCTL not contacted with TMP). In some cases, the number of mCTLs increases after contact with TMP 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 that have not been contacted with TMP.

[0237] Treatment

[0238] The present disclosure provides methods for treating cancer in an individual. The methods generally involve administering to an individual with cancer a composition comprising mCTL of the present disclosure. In some cases, the method comprises: a) introducing to an individual with cancer a composition comprising mCTL and a composition comprising TMP as described herein. In some cases, the method comprises: a) introducing to an individual with cancer a composition comprising mCTL; and b) administering to the individual a TMP as described herein.

[0239] The method of the present disclosure includes administering an effective amount of mCTL. An "effective amount" of mCTL is an amount that, when administered to an individual in need as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy using TMPs and / or immune checkpoint inhibitors, as discussed below) at one or more doses, reduces the overall tumor load of the individual, i.e., the amount of cancer in the body, or keeps the patient's total tumor load relatively stable for a sufficient period of time so that the patient has "stable disease" as confirmed by standard RECIST criteria.

[0240] In some cases, an "effective amount" of mCTLs is an amount that, when administered to an individual in need thereof at one or more doses as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMPs and / or an immune checkpoint inhibitor), results in a sufficient reduction in tumor size, and for a sufficient period of time, for the patient to have a confirmed "partial response" as determined by standard RECIST criteria.

[0241] In some cases, an "effective amount" of mCTLs is an amount that, when administered to an individual in need thereof at one or more doses as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with TMPs and / or an immune checkpoint inhibitor), results in a sufficient reduction in tumor size, and for a sufficient period of time, for the patient to have a confirmed "complete response" as determined by standard RECIST criteria.

[0242] In some cases, an "effective amount" of mCTL is an amount that, when administered as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy using TMP and / or an immune checkpoint inhibitor) to an individual in need (an individual with a tumor) at one or more doses, reduces the tumor volume of the individual. For example, in some cases, an "effective amount" of mCTL is an amount that, when administered as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy using TMP and / or an immune checkpoint inhibitor) to an individual in need (an individual with a tumor) at one or more doses, reduces the tumor volume of 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 of the individual before the administration of mCTL or without the administration of TMP. The tumor volume is determined using the formula (length x width x width) / 2, where the length represents the maximum tumor diameter and the width represents the perpendicular tumor diameter.

[0243] In some cases, an "effective amount" of mCTL is an amount that, when administered to an individual in need at one or more doses, increases the overall survival of the individual. For example, in some cases, an "effective amount" of mCTL is an amount that, when administered to an individual in need at one or more doses as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy using TMP and / or an immune checkpoint inhibitor), increases 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 time of the individual without the administration of TMP.

[0244] In some cases, an "effective amount" of mCTL is an amount that, when administered to an individual in need thereof at one or more doses as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy using TMPs and / or immune checkpoint inhibitors), reduces the level of circulating tumor DNA ("ctDNA") in the 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 level of ctDNA in the individual before administration of mCTL or without administration of TMPs. Any known method can be used to determine ctDNA levels; see, e.g., Cescon et al. (2020) Nature Cancer 1:276.

[0245] In some cases, the methods of the present disclosure include: a) contacting mCTL with TMP in vitro; and b) administering an effective amount of a composition comprising a mixture of mCTL and TMP to an individual in need thereof. In some cases, the contacting step is performed for a period of time to allow proliferation of mCTL in vitro. For example, in some cases, the contacting step is performed for a period of time 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).

[0246] In some cases, the methods of the present disclosure include: 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 cases, TMP is administered prior to administering mCTL. In some cases, TMP is administered both prior to and after administering mCTL. In some cases, TMP is administered both prior to and after administering mCTL.

[0247] In some cases, TMP is administered after mCTL is administered, for example, about 1 hour to about 1 week after the mCTL administration step. For example, in some cases, step (b) is performed within a time period of 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). Thereafter, TMP may be re-administered regularly according to the patient's doctor's determination to maintain the patient's desired mCTL level. For example, TMP may be administered weekly, every two weeks, every three weeks, monthly, or less than monthly frequency. After the initial administration of mCTL, the frequency of administration of TMP may also be higher, and then the frequency of administration may be reduced after the desired mCTL cell level is reached.

[0248] In some cases, the methods of the present disclosure include 10 cells / kg body weight to 10 9 For example, in some cases, the methods of the present disclosure include administering mCTLs in an amount of 10 cells / kg body weight to 10 2 cells / kg body weight, 10 2 cells / kg body weight to 10 3 cells / kg body weight, 10 3 cells / kg body weight to 10 4 cells / kg body weight, 10 4 cells / kg body weight to 10 5 cells / kg body weight, 10 5 cells / kg body weight to 10 6 cells / kg body weight, 10 6 cells / kg body weight to 10 7 cells / kg body weight, 10 7 cells / kg body weight to 10 8 cells / kg body weight, 10 8 cells / kg body weight to 10 9 In some cases, the amount of mCTL is equal to or less than a number selected from the group consisting of: 10 cells / kg body weight, 10 2 cells / kg body weight, 10 3 cells / kg body weight, 10 4 cells / kg body weight, 10 5 cells / kg body weight, 10 6 cells / kg body weight, 10 7 cells / kg body weight, 10 8 cells / kg body weight and 10 9cells / kg body weight. In some cases, the method comprises administering a 7 In some cases, the method comprises administering a composition comprising equal to or less than 10 mCTLs. 6 In some cases, the method comprises administering a composition comprising equal to or less than 10 mCTLs. 5 The composition of mCTLs is in the amount of cells / kg body weight.

[0249] When the methods of the present disclosure include administering TMP, TMP can be administered in an amount between 0.1 mg / kg body weight and 20 mg / kg body weight, such as between 0.1 mg / kg body weight and 10 mg / kg body weight, such as between 0.5 mg / kg body weight and 5 mg / kg body weight, between 1 mg / kg body weight and 5 mg / kg body weight; between 5 mg / kg body weight and 10 mg / kg; between 10 mg / kg body weight and 15 mg / kg; between 15 mg / kg body weight and 20 mg / kg body weight, however, dosages exceeding this exemplary range are also contemplated, particularly in light of the above factors. For example, TMP can be administered in an amount of about 1 mg / kg body weight to about 5 mg / kg body weight, about 5 mg / kg body weight to about 10 mg / kg body weight, about 10 mg / kg body weight to about 15 mg / kg body weight, about 15 mg / kg body weight to about 20 mg / kg body weight. If the regimen is a continuous infusion, the range can also be 1 μg to 10 mg per kilogram of body weight per minute.

[0250] When a TMP as described above is employed, for example a homodimer comprising two heterodimers, wherein each heterodimer comprises two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence set forth in SEQ ID NO:5, such homodimers can be administered in an amount of 4 mg / kg body weight or less, such as 3 mg / kg body weight, 2 mg / kg body weight, 1 mg / kg body weight or less than 1 mg / kg body weight, the specific amount being determined by the patient's physician to maintain the patient's desired mCTL level.

[0251] Generally speaking, when a combination therapy of mCTL and TMP is used, the amount of mCTL administered can be at least one order of magnitude lower than the dose in the absence of TMP, i.e., because TMP can increase the amount of mCTL in the patient. In some cases, the amount of mCTL administered can be at least two orders of magnitude lower than the dose in the absence of TMP. In some cases, the amount of mCTL administered can be at least three orders of magnitude lower than the dose in the absence of TMP.

[0252] For example, according to the FDA-approved package insert, CAR-T cells are genetically modified to produce The amount of such CAR-T cells administered in a monotherapy regimen to an individual with a CD19+ cancer (e.g., a B-cell lymphoma, such as a large B-cell lymphoma of follicular lymphoma) is about 2 x 10 per kg body weight. 6 Up to 2x10 CAR-positive live T cells 8 However, in combination therapy with TMP, a smaller number of live CAR-positive T cells can be administered to patients. CAR-T cells, for example, one order of magnitude less, for example, about 2x10 per kg body weight 5 Up to 2x10 CAR-positive live T cells 7 CAR-positive live T cells, about 2x10 per kg body weight 4 Up to 2x10 CAR-positive live T cells 6 CAR-positive live T cells or about 2x10 per kg body weight 3 Up to 2x10 CAR-positive live T cells 5 In this way, when administering The need for lymphocyte depletion prior to CAR-T cell administration can be reduced or substantially eliminated. After CAR-T cells, recombinant IL-2 (such as aldesleukin The need for treatment with ) can also be reduced or substantially eliminated. CAR-T cells can also reduce the amount of One or more adverse side effects associated with CAR-T cells (e.g., cytokine release syndrome and / or neurotoxicity). or ) and future approved CAR-T therapies, similar results and benefits may be achieved by administering a smaller amount of TMP (e.g., an amount of CAR-T cells that is at least one order of magnitude, at least two orders of magnitude, or at least three orders of magnitude less than the amount specified in the FDA-approved package insert).

[0253] In some cases, the methods disclosed herein for treating cancer do not include a lymphocyte depletion regimen. In other words, in some cases, the individual receiving mCTL therapy does not undergo lymphocyte depletion chemotherapy. Lymphocyte depletion chemotherapy may include administration of, for example, cyclophosphamide and fludarabine prior to infusion of T cells (e.g., 2 to 7 days prior to infusion of T cells).

[0254] In some cases, the methods of the present disclosure for treating cancer do not include administering recombinantly produced IL-2, such as aldesleukin, after administering mCTLs.

[0255] In some cases, the methods of the present disclosure for treating cancer do not include (i) a lymphocyte depletion regimen or (ii) administration of recombinantly produced IL-2, such as aldesleukin, after administration of mCTLs.

[0256] An amount of mCTL can be administered to an individual in need via any of a variety of administration routes. For example, a composition comprising mCTL can be administered via an intramuscular, intravenous, peritumoral, or intratumoral administration route. Similarly, when TMP is administered to an individual, TMP can be administered to the individual via any of a variety of administration routes. For example, a composition comprising TMP (or dimerized TMP) can be administered via an intramuscular, intravenous, peritumoral, or intratumoral administration route.

[0257] Combination therapy

[0258] In some cases, the method disclosed herein for treating cancer in an individual comprises: a) administering mCTL to the individual; and b) administering at least one other therapeutic agent or therapeutic treatment to the individual. In some cases, the method disclosed herein for treating cancer in an individual comprises: a) administering mCTL and TMP to the individual; and b) administering at least one other therapeutic agent or therapeutic treatment to the individual. In some cases, the method disclosed herein for treating cancer in an individual comprises: a) administering mCTL and TMP to the individual; b) administering TMP to the individual; and c) administering at least one other therapeutic agent or therapeutic treatment to the individual. Suitable other therapeutic agents include, but are not limited to, small molecule cancer chemotherapeutics and immune checkpoint inhibitors. Suitable other therapeutic treatments include, for example, radiation therapy, surgery (e.g., surgical removal of a tumor), and the like.

[0259] The treatment method disclosed herein may include co-administering CART-MP and at least one other therapeutic agent. "Co-administration" means administering CART-MP and at least one other therapeutic agent to an individual (although not necessarily at the same time) to achieve a therapeutic effect, which is the result of administering CART-MP and at least one other therapeutic agent. The administration of CART-MP and at least one other therapeutic agent may be substantially simultaneous, for example, 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 administering at least one other therapeutic agent to an individual. In some cases, CART-MP is administered to an individual who is undergoing treatment with at least one other therapeutic agent or has undergone treatment with at least one other therapeutic agent. The administration of CART-MP may occur at different times and / or at different frequencies.

[0260] For example, the treatment method disclosed herein may include co-administration of CART-MP and immune checkpoint inhibitors (such as antibodies specific to immune checkpoints). "Co-administration" means administering both CART-MP and immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoint polypeptides) to an individual to achieve a therapeutic effect, which is the result of administering both CART-MP and immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoint polypeptides). The administration of CART-MP and immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoint polypeptides) may be substantially simultaneous, for example, 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, within 1 week, within 3 weeks, within 3 weeks, within four weeks, or within one month) after administering immune checkpoint inhibitors (e.g., antibodies specific to immune checkpoint polypeptides). In some cases, the CART-MP of the present disclosure is administered to an individual who is undergoing treatment with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) or has undergone treatment with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). Administration of CART-MP and immune checkpoint inhibitors (e.g., antibodies specific for immune checkpoint polypeptides) may occur at different times and / or at different frequencies. If the checkpoint inhibitor has an established dosing interval, the CART-MP may be administered on the same day as the checkpoint inhibitor, depending on the interval. For example, in some cases, where the dosing schedule for pembrolizumab is once every three weeks, a pharmaceutical composition comprising a CART-MP may be administered on the same day.

[0261] Exemplary immune checkpoint inhibitors include targeted immune checkpoint polypeptides, such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, 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 inhibitors. In some cases, immune checkpoint polypeptides are selected from the following stimulating checkpoint molecules: CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from the group consisting of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0262] In some cases, immune checkpoint inhibitors are antibodies specific to immune checkpoint polypeptides. In some cases, anti-immune checkpoint antibodies are monoclonal antibodies. In some cases, anti-immune checkpoint antibodies are humanized, or deimmunized, so that the antibody does not substantially cause an immune response in the human body. In some cases, anti-immune checkpoint antibodies are humanized monoclonal antibodies. In some cases, anti-immune checkpoint antibodies are deimmunized monoclonal antibodies. In some cases, anti-immune checkpoint antibodies are fully human monoclonal antibodies. In some cases, anti-immune checkpoint antibodies inhibit the binding of immune checkpoint polypeptides to the ligands of immune checkpoint polypeptides. In some cases, anti-immune checkpoint antibodies inhibit the binding of immune checkpoint polypeptides to the receptors of immune checkpoint polypeptides.

[0263] 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 HakkoKirin), Varlilumab (CelIDex Therapeutics), Avelumab (EMDSerono), Galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre 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 cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab or PDR001. Suitable anti-PD1 antibodies are described in U.S. Patent Publication No. 2017 / 0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J.Immunother.34:409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some cases, 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 cases, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035 or MSB0010718C. In some cases, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO 2011 / 066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149. In some cases, the immune checkpoint inhibitor is an anti-TIGIT antibody that can bind to a T cell immune receptor (TIGIT) having an immunoglobulin and ITIM domain. In some cases, the anti-TIGIT antibody is BMS-986207 (Bristol-Myers Squibb). In some cases, the anti-TIGIT antibody is tiragolumab. In some cases, the anti-TIGIT antibody is EOS88448 (EOS-448). See, e.g., USPN 11,008,390 and USPN 10,189,902; U.S. Patent Publication No. 2017 / 0088613; and WO 2019 / 137541.

[0264] Among such checkpoint inhibitors, antibodies against PD-1, PD-L1, TIGIT, LAG3, and CTLA-4 are the most common, of which at least nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tisleizumab, and durvalumab have been approved by the FDA and / or regulatory agencies outside the U.S. A certain amount of mCTLs disclosed herein may also be co-administered with a combination of checkpoint inhibitors (e.g., a combination of (i) an antibody against PD-1 or PD-L1 and (ii) an antibody against CTLA-4).

[0265] Subjects suitable for treatment

[0266] Subjects suitable for treatment with the methods of the present disclosure include individuals with cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have failed to respond to treatment, and individuals who have been treated for cancer and initially responded but subsequently became refractory to treatment. Subjects suitable for treatment include individuals with cancers in which cancer cells express or overexpress cancer associated antigens.

[0267] In some cases, the subject is an individual who is undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual who has been treated with an immune checkpoint inhibitor but whose disease has progressed despite such treatment. In some cases, the subject is an individual who is undergoing treatment with a cancer chemotherapeutic agent or has been treated with a cancer chemotherapeutic agent. In some cases, the subject is an individual who is preparing to undergo treatment with an immune checkpoint inhibitor, is undergoing treatment with an immune checkpoint inhibitor, or has been treated with an immune checkpoint inhibitor. In some cases, the subject is preparing to undergo treatment with a cancer chemotherapeutic agent, radiotherapy, surgery, and / or another therapeutic agent; is undergoing treatment with a cancer chemotherapeutic agent, radiotherapy, surgery, and / or another therapeutic agent; or has been treated with a cancer chemotherapeutic agent, radiotherapy, surgery, and / or another therapeutic agent.

[0268] Examples of non-limiting aspects of the present disclosure

[0269] The aspects of the subject matter described above (including embodiments) may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to those skilled in the art after reading this disclosure, each of the individually numbered aspects may be used or combined with any of the previously or later 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:

[0270] Aspect Set 1

[0271] Aspect 1. A quantity of modified cytotoxic T cells ("mCTL"), wherein the mCTL comprises: a) one or more nucleic acids comprising a nucleotide sequence encoding a T cell receptor (TCR) specific for an MHC class I polypeptide, wherein the MHC class I polypeptide presents a protein comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2) ("HPVE7 11-19 / 20TCR" human papillomavirus (HPV) E7 peptide; and b) one or more nucleic acids comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, and wherein the percentage of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition.

[0272] Aspect 2. A certain amount of modified cytotoxic T cells ("mCTL"), wherein the mCTL comprises: a) HPVE7 11-19 / 20 TCR; and b) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, and wherein the percentage of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition.

[0273] Aspect 3. A quantity of modified cytotoxic T cells ("mCTL"), wherein the mCTL comprises: a) a heterologous T cell receptor which is HPV E7 11-19 / 20 TCR; and b) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen.

[0274] Aspect 4. An amount of mCTL according to any one of aspects 1 to 3, wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

[0275] Aspect 5. A certain amount of mCTL according to aspect 4, wherein the cytoplasmic domain comprises one or more co-stimulatory polypeptides.

[0276] Aspect 6. The amount of mCTL according to Aspect 5, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB and OX-40.

[0277] Aspect 7. The composition of any one of Aspects 4 to 6, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0278] Aspect 8. A quantity of mCTL according to any one of aspects 1 to 7, wherein the antigen binding domain is a single-chain Fv polypeptide or a nanobody.

[0279] Aspect 9. An amount of mCTL according to any one of aspects 1 to 8, wherein the CAR is a single polypeptide chain CAR.

[0280] Aspect 10. A quantity of mCTLs according to any one of aspects 1 to 8, wherein the CAR comprises at least two polypeptide chains.

[0281] Aspect 11. An amount of mCTL 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.

[0282] Aspect 12. A certain amount of mCTL according to any one of aspects 1 to 11, wherein the TCR comprises: (i) an α chain, which is Figure 2A The α chain amino acid sequence shown in has at least 90% amino acid sequence identity; and (ii) a β chain, which has Figure 2B The β-chain amino acid sequences shown in have at least 90% amino acid sequence identity.

[0283] Aspect 13. A quantity of mCTLs according to any one of aspects 1 to 11, wherein the TCR comprises: (i) an α chain, which is Figure 2C The α chain amino acid sequence shown in has at least 90% amino acid sequence identity; and (ii) a β chain, which has Figure 2D The β-chain amino acid sequences shown in have at least 90% amino acid sequence identity.

[0284] Aspect 14. An amount of mCTLs according to any one of aspects 1 to 13, wherein the mCTLs are CD8+ T cells.

[0285] Aspect 15. An 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.

[0286] Aspect 16. A composition comprising an amount of T cells, wherein the amount of T cells comprises mCTLs according to any one of aspects 1 to 15, and wherein the percentage of 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%,

[0287] Optionally wherein the composition is a pharmaceutical composition suitable for administration to a human subject.

[0288] Aspect 17. A mixture comprising

[0289] (i) an amount of mCTL according to any one of aspects 1 to 15 or a composition according to aspect 16; and

[0290] (ii) a quantity of a homodimer comprising two heterodimers, wherein each heterodimer comprises:

[0291] a) a first polypeptide comprising:

[0292] i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); and

[0293] ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide, comprising Figure 4B The amino acid sequence shown in , and

[0294] b) a second polypeptide comprising:

[0295] i) two copies of an IL-2 polypeptide, each copy comprising Figure 3B The amino acid sequence shown in ;

[0296] iii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide comprising: Figure 3C The amino acid sequence shown in ; and

[0297] iv) immunoglobulin (Ig) Fc polypeptide,

[0298] wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond.

[0299] Aspect 18. The mixture according to aspect 17,

[0300] wherein the Ig Fc polypeptide comprises Figure 3D The amino acid sequence shown in has at least about 95% amino acid sequence identity, wherein the percentage sequence identity can be determined by sequence alignment using BLAST, and wherein the Ig Fc polypeptide comprises an amino acid sequence based on Figure 3D , Leu at position 14 and Leu at position 15 according to the amino acid numbering shown in .

[0301] wherein the first polypeptide and the second polypeptide 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,

[0302] wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide, and wherein the second polypeptide comprises a peptide linker between one or more of:

[0303] a) a first copy of said IL-2 polypeptide and a second copy of said IL-2 polypeptide;

[0304] b) one of the two copies of said IL-2 polypeptide and said MHC class I heavy chain polypeptide; and

[0305] c) said MHC class I heavy chain polypeptide and said Ig Fc polypeptide,

[0306] Optionally, the homodimer comprises two heterodimers, wherein each heterodimer comprises a first polypeptide comprising the amino acid sequence shown in SEQ ID NO:14, and a second polypeptide comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:18.

[0307] Aspect 19. A method for preparing a certain amount of mCTL according to any one of aspects 1 to 15, wherein the method comprises modifying a certain amount of T cells by introducing one or more nucleic acids encoding

[0308] (i) a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a cancer associated antigen, and

[0309] (ii) HPV E7 11-19 / 20 TCR.

[0310] Aspect 20. The method according to aspect 19, wherein the method comprises at least partially separating the HPVE7 11-19 / 20 The step of at least partially isolating target T cells containing HPV E7 11-19 / 20 The step of targeting T cells with a TCR comprises binding the target T cells to an HPV16 E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2).

[0311] Aspect 21. The method according to aspect 20, wherein the HPV16 E7 peptide is immobilized on an insoluble support.

[0312] Aspect 22. The method according to aspect 21, wherein the insoluble support is a bead.

[0313] Aspect 23. The method according to aspect 19, wherein the HPV16 peptide is a peptide-loaded MHC class I multimer.

[0314] Aspect 24. A method according to any one of Aspects 19 to 23, wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

[0315] Aspect 25. A method according to aspect 24, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0316] Aspect 26. The method according to aspect 24 or 25, wherein the cytoplasmic domain comprises one or more co-stimulatory polypeptides.

[0317] Aspect 27. The method according to aspect 26, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB and OX-40.

[0318] Aspect 28. A method according to any one of Aspects 19 to 27, wherein the antigen binding domain is a single-chain Fv polypeptide or a Nanobody.

[0319] Aspect 29. A method according to any one of Aspects 19 to 28, wherein the CAR is a single polypeptide chain CAR.

[0320] Aspect 30. A method according to any one of Aspects 19 to 28, wherein the CAR comprises two polypeptide chains.

[0321] Aspect 31. A method according to 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.

[0322] Aspect 32. The method of any one of Aspects 19 to 31, wherein the percentage of the total number of T cells in the 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%.

[0323] Aspect 33. The method according to any one of aspects 19 to 32, wherein prior to step (ii), the composition comprising a quantity of T cells is contacted in vitro or in vivo with a TMP, wherein the TMP comprises a heterodimeric polypeptide comprising:

[0324] 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; and

[0325] 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,

[0326] Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, wherein each heterodimeric polypeptide comprises a first polypeptide comprising the amino acid sequence shown in SEQ ID NO:14, and a second polypeptide comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:18.

[0327] Aspect 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.

[0328] Aspect 35. The method according to any one of aspects 19 to 34, comprising enriching the T cells for CD8+ T cells between steps (i) and (ii).

[0329] Aspect 36. The method according to any one of aspects 19 to 35, comprising enriching the T cells for CD8+ T cells between steps (ii) and (iii).

[0330] Aspect 37. A method of treating cancer in an individual, the method comprising introducing into the individual a composition comprising an amount of a modified cytotoxic T cell according to any one of aspects 1 to 15 or a pharmaceutical composition prepared according to any one of aspects 16 to 33.

[0331] Aspect 38. The method according to aspect 37, further comprising administering to the individual a composition comprising TMP, wherein the TMP comprises any of the following:

[0332] a) a heterodimeric polypeptide comprising

[0333] 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; and

[0334] a second polypeptide comprising: (i) two copies of an IL-2 polypeptide; (ii) an MHC class I heavy chain polypeptide; and (iii) a variant immunoglobulin (Ig) Fc polypeptide,

[0335] Optionally wherein the TMP comprises a homodimer comprising two heterodimeric polypeptides, wherein each heterodimeric polypeptide comprises 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;

[0336] or

[0337] 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.

[0338] Aspect 39. A method according to Aspect 37 or Aspect 38, wherein the administration of a composition comprising an amount of genetically modified cytotoxic T cells comprises administering an amount of genetically modified cytotoxic T cells that is equal to or less than a number selected from the group consisting of: 10 cells / kg body weight, 102 cells / kg body weight, 103 cells / kg body weight, 104 cells / kg body weight, 105 cells / kg body weight, 106 cells / kg body weight, 107 cells / kg body weight, 108 cells / kg body weight and 109 cells / kg body weight.

[0339] Aspect 40. The method according to any one of Aspects 37 to 39, wherein the administering of a composition comprising an amount of genetically modified cytotoxic T cells comprises administering an amount of genetically modified cytotoxic T cells that is equal to or less than 107 cells / kg body weight.

[0340] Aspect 41. The method according to any one of aspects 37 to 40, wherein the individual has not undergone a lymphodepleting regimen prior to said introducing step.

[0341] Aspect 42. The method according to any one of Aspects 37 to 41, wherein the administration is intramuscular, intravenous, peritumoral or intratumoral administration.

[0342] Aspect 43. The method according to any one of Aspects 37 to 42, further comprising administering one or more checkpoint inhibitors to the individual.

[0343] Aspect 44. A method according to Aspect 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, PI3Kδ, PI3Kγ, 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.

[0344] Aspect 45. A method according to Aspect 43, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1 or CTLA4.

[0345] Aspect 46. The method of aspect 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, avelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lilizumab, usileumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogazumab, vanlilumab, avelumab, galiximab, AMP-514, AUNP 12, indomod, NLG-919, INCB024360, KN035, and combinations thereof.

[0346] Aspect 47. A quantity of modified cells according to any one of aspects 1 to 15, wherein the HPV E7 11-19 / 20TCR, the modified cell comprises 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.

[0347] Aspect 48. A quantity of modified cells according to any one of aspects 1 to 15 or 47, wherein instead of CAR, the modified cells comprise an antigen binding domain other than CAR.

[0348] Aspect 48. An amount of modified cells according to any one of aspects 1 to 15, 47 or 48, wherein instead of T cells, the modified cells are cells other than T cells, such as NK cells.

[0349] Aspect Set 2

[0350] Aspect 1. A certain amount of modified cytotoxic cells ("mCL"), wherein the mCL comprises: a) one or more nucleic acids comprising a nucleotide sequence encoding a predetermined T cell receptor (TCR); and b) an antigen binding domain specific for a cancer-associated antigen, and wherein the percentage of target mCL cells in the composition exceeds at least 1% of the total number of T cells in the composition.

[0351] Aspect 2. A quantity of modified cytotoxic cells ("mCL"), wherein the mCL comprises: a) HPV E7 11-19 / 20 TCR; and b) an antigen binding domain specific for a cancer associated antigen, and wherein the percentage of target mCL cells in the composition exceeds at least 1% of the total number of T cells in the composition, optionally wherein the antigen binding domain is in a heterologous antigen binding domain.

[0352] Aspect 3. A quantity of modified cytotoxic cells ("mCL"), wherein the mCL comprises: a) a heterologous T cell receptor which is HPV E7 11-19 / 20 TCR; and b) a heterologous antigen binding domain specific for a cancer associated antigen.

[0353] Aspect 4. An amount of mCL according to any one of Aspects 1 to 3, wherein the mCL comprises a CAR, and wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

[0354] Aspect 5. A quantity of mCL according to Aspect 4, wherein the cytoplasmic domain comprises one or more co-stimulatory polypeptides.

[0355] Aspect 6. The amount of mCL according to Aspect 5, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB and OX-40.

[0356] Aspect 7. The composition of any one of Aspects 4 to 6, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0357] Aspect 8. An 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.

[0358] Aspect 9. An amount of mCL according to any one of aspects 1 to 8, wherein the mCL comprises a CAR, and wherein the CAR is a single polypeptide chain CAR.

[0359] Aspect 10. An amount of mCL according to any one of aspects 1 to 8, wherein the mCL comprises a CAR, and wherein the CAR comprises at least two polypeptide chains.

[0360] Aspect 11. An 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.

[0361] Aspect 12. An amount of mCL according to any one of aspects 1 to 11, wherein the TCR comprises: (i) an α chain, which is Figure 2A The α chain amino acid sequence shown in has at least 90% amino acid sequence identity; and (ii) a β chain, which has Figure 2B The β-chain amino acid sequences shown in have at least 90% amino acid sequence identity.

[0362] Aspect 13. An amount of mCL according to any one of aspects 1 to 11, wherein the TCR comprises: (i) an α chain, which is Figure 2C The α chain amino acid sequence shown in has at least 90% amino acid sequence identity; and (ii) a β chain, which has Figure 2D The β-chain amino acid sequences shown in have at least 90% amino acid sequence identity.

[0363] Aspect 14. An amount of mCL according to any one of aspects 1 to 13, wherein the mCL is a NK cell, a macrophage or an ILC (innate lymphoid cell), and wherein the cell has a native, modified or heterologous antigen binding domain.

[0364] Aspect 15. An amount of mCL according to any one of Aspects 1 to 13, wherein the mCL is a T cell, a classical T cell (expressing αβ receptors), a T cell expressing γ / δ TCR, a CD8 phenotype and a CD4 phenotype, a MAIT T cell restricted to MR-1 non-classical HLA, a killer T cell or a killer innate-like T cell.

[0365] Aspect 16. A composition comprising an amount of cells, wherein the amount of cells comprises mCL according to any one of aspects 1 to 15, and wherein the percentage of cells that are mCL 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%,

[0366] Optionally wherein the composition is a pharmaceutical composition suitable for administration to a human subject.

[0367] Aspect 17. A mixture comprising

[0368] (i) an amount of mCL according to any one of aspects 1 to 15 or a composition according to aspect 16; and

[0369] (ii) a quantity of a homodimer comprising two heterodimers, wherein each heterodimer comprises:

[0370] a) a first polypeptide comprising:

[0371] i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); and

[0372] ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide, comprising Figure 4B The amino acid sequence shown in , and

[0373] b) a second polypeptide comprising:

[0374] i) two copies of an IL-2 polypeptide, each copy comprising Figure 3B The amino acid sequence shown in ;

[0375] iii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide comprising: Figure 3C The amino acid sequence shown in ; and

[0376] iv) immunoglobulin (Ig) Fc polypeptide,

[0377] wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond.

[0378] Aspect 18. The mixture according to aspect 17,

[0379] wherein the Ig Fc polypeptide comprises Figure 3D The amino acid sequence shown in has at least about 95% amino acid sequence identity, wherein the percentage sequence identity can be determined by sequence alignment using BLAST, and wherein the Ig Fc polypeptide comprises an amino acid sequence based on Figure 3D , Leu at position 14 and Leu at position 15 according to the amino acid numbering shown in .

[0380] wherein the first polypeptide and the second polypeptide 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,

[0381] wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide, and wherein the second polypeptide comprises a peptide linker between one or more of:

[0382] a) a first copy of said IL-2 polypeptide and a second copy of said IL-2 polypeptide;

[0383] b) one of the two copies of said IL-2 polypeptide and said MHC class I heavy chain polypeptide; and

[0384] c) said MHC class I heavy chain polypeptide and said Ig Fc polypeptide,

[0385] Optionally, the homodimer comprises two heterodimers, wherein each heterodimer comprises a first polypeptide comprising the amino acid sequence shown in SEQ ID NO:14, and a second polypeptide comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:18.

[0386] Aspect 19. A method of producing a quantity of mCL according to any one of aspects 1 to 15, wherein the method comprises modifying a quantity of cells by introducing one or more nucleic acids encoding

[0387] (i) an antigen binding domain specific for a cancer associated antigen, and

[0388] (ii) HPV E7 11-19 / 20 TCR.

[0389] Aspect 20. The method according to aspect 19, wherein the method comprises at least partially separating the HPVE7 11-19 / 20 a step of at least partially isolating target cells containing HPV E7 11-19 / 20 The step of targeting cells with a TCR comprises binding the target cells to an HPV16 E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2).

[0390] Aspect 21. The method according to aspect 20, wherein the HPV16 E7 peptide is immobilized on an insoluble support.

[0391] Aspect 22. The method according to aspect 21, wherein the insoluble support is a bead.

[0392] Aspect 23. The method according to aspect 19, wherein the HPV16 peptide is a peptide-loaded MHC class I multimer.

[0393] Aspect 24. A method according to any one of Aspects 19 to 23, wherein the mCL comprises a CAR, and wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

[0394] Aspect 25. A method according to aspect 24, wherein the intracellular signaling domain comprises a signaling domain from the zeta chain of human CD3.

[0395] Aspect 26. The method according to aspect 24 or 25, wherein the cytoplasmic domain comprises one or more co-stimulatory polypeptides.

[0396] Aspect 27. The method according to aspect 26, wherein the co-stimulatory polypeptide is selected from CD28, 4-1BB and OX-40.

[0397] Aspect 28. A method according to any one of Aspects 19 to 27, wherein the antigen binding domain is a single-chain Fv polypeptide or a Nanobody.

[0398] Aspect 29. A method according to any one of Aspects 19 to 28, wherein mCL comprises a CAR, and wherein the CAR is a single polypeptide chain CAR.

[0399] Aspect 30. A method according to any one of aspects 19 to 28, wherein mCL comprises a CAR, and wherein the CAR comprises two polypeptide chains.

[0400] Aspect 31. A method according to 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.

[0401] Aspect 32. A method according to any one of Aspects 19 to 31, wherein the percentage of the total number of cells in the composition that are target mCL 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%.

[0402] Aspect 33. The method according to any one of aspects 19 to 32, wherein prior to step (ii), the composition comprising a quantity of cells is contacted in vitro or in vivo with a TMP, wherein the TMP comprises a heterodimeric polypeptide comprising:

[0403] 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; and

[0404] 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,

[0405] Optionally, the TMP comprises a homodimer comprising two heterodimeric polypeptides, wherein each heterodimeric polypeptide comprises a first polypeptide comprising the amino acid sequence shown in SEQ ID NO:14, and a second polypeptide comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:18.

[0406] Aspect 34. The method according to any one of aspects 19 to 33, wherein at least 50% of the target cells are CD8+ T cells.

[0407] Aspect 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).

[0408] Aspect 36. A method according to any one of aspects 19 to 35, comprising enriching the cells for CD8+ T cells between steps (ii) and (iii).

[0409] Aspect 37. A method of treating cancer in an individual, the method comprising introducing into the individual a composition comprising an amount of a modified cytotoxic cell according to any one of aspects 1 to 15 or a pharmaceutical composition prepared according to any one of aspects 16 to 33.

[0410] Aspect 38. The method according to aspect 37, further comprising administering to the individual a composition comprising TMP, wherein the TMP comprises any of the following:

[0411] a) a heterodimeric polypeptide comprising

[0412] 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; and

[0413] a second polypeptide comprising: (i) two copies of an IL-2 polypeptide; (ii) an MHC class I heavy chain polypeptide; and (iii) a variant immunoglobulin (Ig) Fc polypeptide,

[0414] Optionally wherein the TMP comprises a homodimer comprising two heterodimeric polypeptides, wherein each heterodimeric polypeptide comprises 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;

[0415] or

[0416] 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.

[0417] Aspect 39. The method of aspect 37 or aspect 38, wherein administering a composition comprising an amount of genetically modified cytotoxic cells comprises administering an amount of genetically modified cytotoxic cells equal to or less than a number selected from the group consisting of: 10 cells / kg body weight, 10 2 cells / kg body weight, 10 3 cells / kg body weight, 10 4 cells / kg body weight, 10 5 cells / kg body weight, 10 6 cells / kg body weight, 10 7 cells / kg body weight, 10 8 cells / kg body weight and 10 9 cells / kg body weight.

[0418] Aspect 40. The method according to any one of aspects 37 to 39, wherein administering a composition comprising an amount of genetically modified cytotoxic cells comprises administering an amount of genetically modified cytotoxic cells that is equal to or less than 10 7 cells / kg body weight.

[0419] Aspect 41. The method according to any one of aspects 37 to 40, wherein the individual has not undergone a lymphodepleting regimen prior to said introducing step.

[0420] Aspect 42. The method according to any one of Aspects 37 to 41, wherein the administration is intramuscular, intravenous, peritumoral or intratumoral administration.

[0421] Aspect 43. The method according to any one of Aspects 37 to 42, further comprising administering one or more checkpoint inhibitors to the individual.

[0422] Aspect 44. A method according to Aspect 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, PI3Kδ, PI3Kγ, 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.

[0423] Aspect 45. A method according to Aspect 43, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1 or CTLA4.

[0424] Aspect 46. The method of aspect 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, avelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lilizumab, usileumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogazumab, vanlilumab, avelumab, galiximab, AMP-514, AUNP 12, indomod, NLG-919, INCB024360, KN035, and combinations thereof.

[0425] Example

[0426] The following examples are presented to provide a more complete disclosure to those of ordinary skill in the art and are not intended to limit the scope of what the inventors regard as their invention, nor do they represent that the experiments below are all or the only experiments performed.

[0427] Example 1

[0428] By adding the encoding HPV E7 11-19 / 20 One or more nucleic acids encoding TCR are used to modify the CAR-T cell population in vitro to prepare a composition of mCTL. Alternatively, first, a nucleic acid encoding HPV E7 is added 11-19 / 20 The T cells are modified by adding a nucleic acid encoding a TCR, and then the T cells are modified by adding one or more nucleic acids encoding a CAR to prepare a composition comprising a population of mCTLs. For example, recombinantly produced IL-2 (such as ) or TMP (such as CUE-101), to increase the mCTL population in vitro.

[0429] Prior to administration of the mCTL, the patient did not receive lymphodepleting therapy. The amount of mCTL administered to the patient was at least an order of magnitude lower than the dose that would have been administered had TMP not been administered. After administration of the mCTL, one or more doses of CUE-101 were administered to the patient in an amount up to about 4 mg / kg body weight to increase the amount of mCTL to a desired range, and then maintain the amount of mCTL within the desired range. After administration of the mCTL, the patient did not receive wild-type IL-2, such as After administration of mCTLs, the patient did not experience severe symptoms associated with cytokine release syndrome.

[0430] Example 2

[0431] Example 2 is performed identically to Example 1, except that a dose of CUE-101 is administered to the patient in an amount up to about 4 mg / kg body weight prior to administration of mCTL. After administration of mCTL, one or more additional doses of CUE-101 are administered to the patient in an amount up to about 4 mg / kg body weight to increase the amount of mCTL to a desired range and / or to maintain the amount of mCTL within a desired range. After administration of mCTL, the patient does not receive wild-type IL-2, such as After administration of mCTLs, the patient did not experience severe symptoms associated with cytokine release syndrome.

[0432] Example 3

[0433] A composition of mCTL was prepared, which contained a protein encoding HPV E7 11-19 / 20 TCR nucleic acid CAR-T cells.

[0434] The patient has not received lymphodepleting therapy prior to administration of the mCTL. Optionally, a dose of CUE-101 is administered to the patient in an amount up to about 4 mg / kg body weight prior to administration of the mCTL to the patient.

[0435] Administered to patients The amount of CAR-T is up to about 2x10 per kg body weight. 5 Up to 2x10 CAR-positive live T cells 7 CAR-positive live T cells, optionally up to about 2 x 10 per kg body weight 4 Up to 2x10 CAR-positive live T cells 6CAR-positive live T cells. After administration of mCTL, additional doses of CUE-101 are administered to the patient in an amount up to about 4 mg / kg body weight to increase the amount of mCTL to the desired range, and then the amount of mCTL is maintained within the desired range. After administration of mCTL, the patient does not receive wild-type IL-2, such as After administration of mCTLs, the patient did not experience severe symptoms associated with cytokine release syndrome.

[0436] Although the present disclosure has been described with reference to specific embodiments of the present disclosure, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications may be made to adapt specific circumstances, materials, material compositions, processes, process steps or multiple steps to the purpose, 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 ("mCTL"), wherein the mCTL comprises: 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 a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, and wherein the percentage of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition.

2. A certain amount of modified cytotoxic T cells ("mCTL"), wherein the mCTL comprises: a) 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) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen, and wherein the percentage of target mCTL cells in the composition exceeds at least 1% of the total number of T cells in the composition.

3. A quantity of modified cytotoxic T cells ("mCTL"), wherein the mCTL comprises: a) a heterologous 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) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cancer associated antigen.

4. The amount of mCTL according to any one of claims 1 to 3, wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

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

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

7. The composition of any one of claims 4 to 6, 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 7, wherein the antigen binding domain is a single-chain Fv polypeptide or a nanobody.

9. The amount of mCTL according to any one of claims 1 to 8, wherein the CAR is a single polypeptide chain CAR.

10. The amount of mCTL according to any one of claims 1 to 8, wherein the CAR comprises at least two polypeptide chains.

11. The amount of mCTL according to any one of claims 1 to 10, wherein the cancer-associated antigen is selected from the group consisting of 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 amount of mCTL according to any one of claims 1 to 11, wherein the TCR comprises: (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG2A ; and (ii) a β chain having at least 90% amino acid sequence identity with the β chain amino acid sequence shown in FIG2B .

13. The amount of mCTL according to any one of claims 1 to 11, wherein the TCR comprises: (i) an α chain having at least 90% amino acid sequence identity with the α chain amino acid sequence shown in FIG. 2C ; and (ii) a β chain having at least 90% amino acid sequence identity with the β chain amino acid sequence shown in FIG. 2D .

14. The amount of mCTL according to any one of claims 1 to 13, wherein the mCTL is a CD8+ T cell.

15. A composition comprising an amount of T cells, wherein the amount of T cells comprises the mCTL according to any one of claims 1 to 13, and wherein the percentage of 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%, 16. The composition of claim 15, wherein the composition is a pharmaceutical composition suitable for administration to a human subject.

17. A mixture comprising (i) an amount of mCTL according to any one of claims 1 to 14 or a composition according to claim 15 or 16; and (ii) a quantity of a homodimer comprising two heterodimers, wherein each heterodimer comprises: a) a first polypeptide comprising: i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2); and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide comprising the amino acid sequence as shown in FIG. 4B , and b) a second polypeptide comprising: i) two copies of an IL-2 polypeptide, each copy comprising the amino acid sequence as shown in FIG3B ; iii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide comprising the amino acid sequence as shown in Figure 3C; and iv) immunoglobulin (Ig) Fc polypeptide, wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond.

18. The mixture according to claim 17, wherein 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 Figure 3D, wherein the percent sequence identity can be determined by sequence alignment using BLAST, and wherein the Ig Fc polypeptide comprises Leu at position 14 and Leu at position 15 based on the amino acid numbering shown in Figure 3D. wherein the first polypeptide and the second polypeptide 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, wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide, and wherein the second polypeptide comprises a peptide linker between one or more of: 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.

19. A method for preparing a certain amount of mCTL according to any one of claims 1 to 14, wherein the method comprises modifying a certain amount of T cells by introducing one or more nucleic acids encoding (i) a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a cancer associated antigen, and (ii)HPV E7 11-19 / 20 TCR。 20. The method of claim 19, wherein the method comprises at least partially isolating a polypeptide comprising HPV E7 11-19 / 20 The step of at least partially isolating target T cells containing HPV E7 11-19 / 20 The step of targeting T cells with a TCR comprises binding the target T cells to an HPV16 E7 peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 1) or YMLDLQPET (SEQ ID NO: 2).

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

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

23. The method of claim 19, wherein the HPV16 E7 peptide is a peptide-loaded MHC class I multimer.

24. The method of any one of claims 19 to 23, wherein the CAR comprises: a) an extracellular domain comprising the antigen binding domain; b) a transmembrane region; and c) a cytoplasmic domain comprising an intracellular signaling domain.

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

26. The method of claim 24 or 25, wherein the cytoplasmic domain comprises one or more co-stimulatory polypeptides.

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

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

29. The method of any one of claims 19 to 28, wherein the CAR is a single polypeptide chain CAR.

30. The method of any one of claims 19 to 28, wherein the CAR comprises two polypeptide chains.

31. The method of any one of claims 19 to 30, wherein the cancer-associated antigen is selected from the group consisting of 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, optionally wherein the CAR comprises a scFv that binds CD19 or BCMA.

32. The method of any one of claims 19 to 31, wherein the percentage 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. The method of any one of claims 19 to 32, wherein prior to step (ii), the composition comprising an amount of T cells is contacted in vitro or in vivo with a composition comprising a T cell modulating polypeptide (TMP), the T cell modulating polypeptide comprising: 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; 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.

34. The method of any one of claims 19 to 33, wherein at least 50% of the target T cells are CD8+ T cells.

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

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

37. A method of treating cancer in an individual, the method comprising introducing into the individual a composition comprising an amount of a modified cytotoxic T cell according to any one of claims 1 to 15 or a pharmaceutical composition prepared by the method of any one of claims 16 to 33.

38. The method of claim 37, further comprising administering to the individual a composition comprising TMP, the TMP comprising: 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; 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, Optionally, the homodimer comprises two heterodimers, wherein each heterodimer comprises a first polypeptide comprising the amino acid sequence shown in SEQ ID NO:14, and a second polypeptide comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:

18.

39. The method of claim 37 or claim 38, wherein the administration of a composition comprising an amount of genetically modified cytotoxic T cells comprises administering an amount of genetically modified cytotoxic T cells equal to or less than an amount selected from the group consisting of: 10 cells / kg body weight, 10 2 cells / kg body weight, 10 3 cells / kg body weight, 10 4 cells / kg body weight, 10 5 cells / kg body weight, 10 6 cells / kg body weight, 10 7 cells / kg body weight, 10 8 cells / kg body weight and 109 cells / kg body weight.

40. The method of any one of claims 37 to 39, wherein the administration of a composition comprising an amount of genetically modified cytotoxic T cells comprises administering an amount of genetically modified cytotoxic T cells that is equal to or less than 10 7 cells / kg body weight.

41. The method of any one of claims 37 to 40, wherein the individual has not undergone a lymphodepleting regimen prior to the introducing step.

42. The method of any one of claims 37 to 41, wherein the administration is intramuscular, intravenous, peritumoral or intratumoral administration.

43. The method of any one of claims 37 to 42, further comprising administering one or more checkpoint inhibitors to the individual.

44. The method of 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, PI3Kδ, PI3Kγ, 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 of claim 43, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1, or CTLA4.

46. ​​The method of claim 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, avelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lilizumab, usileumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogazumab, vanlilumab, avelumab, galiximab, AMP-514, AUNP 12, indomod, NLG-919, INCB024360, KN035, and combinations thereof.

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