Improved T cell manufacturing process

Abstract

The present invention relates to improved methods for producing T cells and improved T cell compositions resulting therefrom.

Description

[Technical Field]

[0001] The present invention relates to improved methods for producing T cells and improved T cell compositions resulting therefrom. The present invention further relates to methods for producing T cells that provide improved T cell expansion and result in T cell populations with improved persistence, memory function, and antigen-stimulated survival. The present invention further relates to the use of improved T cell populations in adoptive T cell immunotherapy, including adoptive therapy, for the treatment of tumors and cancer. [Background technology]

[0002] Adoptive cell therapy (ACT) can involve the intravenous transfer of tumor-resident peripheral blood-modified immune cells into cancer patients to mediate antitumor functions, thereby offering an opportunity to treat diseases including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiencies. ACT may also involve the transfer of tumor-infiltrating lymphocytes (TILs) or natural killer cells (NK cells) as the basis for cellular cancer therapy. In addition, ACT using genetically modified T cells expressing novel T cell receptors (TCRs) or chimeric antigen receptors (CARs) offers the opportunity to provide a large pool of tumor-specific T cells that can be generated with specific and potent antitumor activity to recognize specific tumor-expressed antigens and improve and target clinical responses.

[0003] The ACT process typically involves a step of leukapheresis in which the donor's blood is passed through a device that separates leukocytes from the blood sample. The isolated T cell population is then subjected to a process that includes activation and expansion steps, and optionally genetic modification to introduce specific CAR or TCR molecules and generate the number of activated T cells required for a therapeutic dose to be clinically effective.

[0004] Current T cell manufacturing processes often require multiple rounds of activation and expansion to achieve a T cell population large enough for therapeutic doses. Population expansion is a limiting step, and T cell activation and expansion methods often produce cell populations enriched in a subset of relatively differentiated cells; these cells, by their nature, have limited survival and short lifespans, and therefore provide short-lived and less potent anti-tumor activity and memory functions. Such T cell populations are prone to exhaustion and loss of effector immune cell function, which is a disadvantage for in vivo expansion of transferred T cells, and lack persistence after infusion into patients.

[0005] The present invention addresses the above-mentioned limitations of current processes for T cell manufacturing and therapeutic T cells and provides methods for therapeutic T cell compositions with improved expansion, persistence and survival. Summary of the Invention

[0006] The present invention generally provides improved methods of producing T cells and / or populations of T cells, thereby providing improved populations of T cells and / or compositions of T cells produced by the methods, and the present invention further provides uses of improved populations of T cells and / or compositions of T cells in adoptive therapy for the treatment of diseases including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immune disorders. Methods according to the present invention provide improved T cell expansion, survival, effector and memory function in vitro and / or in vivo.

[0007] According to the present invention, the method comprises the following steps: (a) activating an isolated population of T cells; (b) culturing the T cells; (c) modifying the T cells to express a xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR); (d) adding an inhibitor of AKT (AKT inhibitor) to the modified T cells; (e) culturing the modified T cells or T cell population to expand and / or grow the cells; (f) optionally, recovering and / or cryopreserving the engineered T cells or T cell population, wherein optionally the xenogeneic TCR or CAR binds or specifically binds to a cancer and / or tumor antigen or a peptide antigen thereof; optionally, the T cells are engineered to express a xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR), e.g., by transducing the T cells with a nucleic acid or vector comprising a nucleic acid encoding one or more xenogeneic T cell receptors (TCRs) and / or one or more chimeric antigen receptors (CARs).

[0013] Methods for producing modified T cells and / or populations of modified T cells are provided, comprising:

[0008] T cell adoptive therapy The isolated T cells or population of T cells may be T cells, tumor-infiltrating cytotoxic T lymphocytes (TILs), or natural killer cells (NK cells). T cells may be natural killer T (NKT) cells or their precursors, including embryonic stem cells and pluripotent stem cells (e.g., from which lymphoid cells can differentiate). T cells mature in the thymus and are lymphocytes primarily responsible for cell-mediated immunity, but can also participate in the adaptive immune system. According to the present invention, T cells may include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), or memory T cells, such as TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, or gamma-delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that are capable of inducing the killing of infected somatic or tumor cells. The T cells provided herein in accordance with the invention are CD8 + T cells or CD4 + T cells; or CD4 +T cells and CD8 + For example, the T cell may be a CD4 + T cells and CD8 + It may be a mixed population of T cells. + T cells are T helper cells (T H CD8 T cells, known as T cells, express the CD4 surface glycoprotein and play a key role in the adaptive immune system, assisting the activity of other immune cells by releasing T cell cytokines and helping to suppress or regulate immune responses. They are essential for the activation and development of cytotoxic T cells. + T cells are cytotoxic T cells (T C These are known as CTLs (killer T cells) and express the CD8 surface glycoprotein. + T cells act to destroy virus-infected cells and tumor cells. The majority of CD8 + T cells express a TCR, which can recognize specific antigens presented on the surface of infected or damaged cells by class I MHC molecules. Specific binding of the TCR and optionally the CD8 glycoprotein to the antigen and MHC molecule results in T cell-mediated destruction of the infected or damaged cell.

[0009] The isolated population of T cells can be isolated from a donor subject. According to the present invention, the T cells produced by the method of the present invention can be used for adoptive cell therapy or adoptive immunotherapy. The donor subject and the recipient individual (receiving adoptive cell therapy or adoptive immunotherapy) can be the same (i.e., autologous therapy; T cells are obtained from an individual who will then be treated with modified T cells), or the donor and recipient individuals can be different (i.e., allogeneic therapy; T cells are obtained from one individual and then used to treat a different individual). Autologous refers to any material from a subject that will later be reintroduced into the same subject.

[0010] Thus, adoptive cell therapy or adoptive immunotherapy in the context of the present invention refers to the adoptive transfer of isolated T cells or populations of T cells that have been genetically engineered by gene transfer to express genetically modified TCRs or CARs and / or co-receptors (e.g., CD8) specific for surface antigens, antigenic peptides, or antigenic peptides expressed on target cells, optionally as MHC complexes. This can be used to treat a wide range of diseases depending on the target selected, e.g., tumor- or cancer-specific antigens or antigenic peptides to treat cancer or tumors. Briefly, adoptive cell therapy involves removing a portion of a donor's or patient's white blood cells using a process called leukapheresis. Subsequently, T cells, TILs, or NK cells can be expanded and mixed with an expression vector containing a TCR / CAR polynucleotide and / or co-receptor (e.g., CD8) to transfer the TCR / CAR and / or co-receptor (e.g., CD8) into the T cells, TILs, or NK cells. The T cells, TILs or NK cells are expanded again, and at the end of expansion, the engineered T cells or NK cells can be washed, concentrated and subsequently frozen to allow time for testing, transport and storage until the patient is ready to receive an infusion of the engineered cells.

[0011] cell culture The modified T cells can be cultured using any convenient means, technique, vessel, container, or system to produce an expanded population. Suitable culture systems include stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, particularly hollow fiber bioreactors. Preferably, the use of such systems is well known in the art. Preferably, the culture means is a gas-permeable rapid expansion culture, e.g., G-Rex™, a device for expansion or static expansion of cells, such as non-adherent cells. and / or cells according to the methods of the present invention.

[0012] Cellular modification According to the present invention, T cells are modified to express one or more xenogeneic T cell receptors (TCRs) and / or one or more chimeric antigen receptors (CARs), and the modification may be by transducing the T cells with a nucleic acid or vector comprising a nucleic acid encoding one or more xenogeneic T cell receptors (TCRs) and / or one or more chimeric antigen receptors (CARs). T cells can also be modified by integration of a nucleic acid encoding one or more xenogeneic T cell receptors (TCRs) and / or one or more chimeric antigen receptors (CARs) into the genome of the T cell, e.g., into the genome of a precursor of the T cell, e.g., an induced pluripotent stem cell or a lymphoid lineage cell derived therefrom, e.g., a mature T cell derived therefrom.

[0013] According to the present invention, modified T cells can be modified to contain a heterologous nucleic acid or nucleic acid construct encoding a heterologous T cell receptor (TCR) or a heterologous chimeric antigen receptor (CAR), or a vector comprising the nucleic acid or construct. Optionally, the TCR can be an affinity-enhanced TCR, such as a specific peptide-enhanced affinity receptor (SPEAR) TCR.

[0014] According to the present invention, the method may comprise the inclusion of poloxamer at any stage or step within the process, preferably at the time of modifying or transducing the T cell or T cell population, for the purpose of modifying and / or transducing, optionally at a level of up to or about half the multiplicity of infection (MOI) of the virus relative to cell concentration (i.e., 0.5 virus per cell), optionally at any one of 0.1-0.2, 0.3-0.4, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2.0.

[0015] Heterogeneous TCR / CAR In accordance with the present invention, engineered T cells can express at least one xenogeneic T cell receptor (TCR) and / or xenogeneic chimeric antigen receptor (CAR) that binds or specifically binds to a cancer and / or tumor antigen or peptide antigen thereof, preferably a peptide antigen associated with a cancerous condition, cancer, and / or tumor, and / or presented by a cancer cell or tumor of tissue, optionally presented by HLA / MHC. Upon antigen binding, the engineered T cell or population of T cells can exhibit T cell effector function and / or cytolytic activity against antigen-bearing cells and / or undergo proliferation and / or cell division. In certain embodiments, engineered T cells or populations of T cells comprising a TCR exhibit comparable or better therapeutic efficacy compared to cells comprising a chimeric antigen receptor (CAR) targeting the same cancer and / or tumor antigen and / or peptide (antigenic peptide). Activated modified T cells or populations of T cells containing a heterologous TCR or CAR can secrete anti-tumor cytokines, which may include, but are not limited to, TNFα, IFNγ, and IL2.

[0016] The term "heterologous" or "exogenous" refers to a polypeptide or nucleic acid that is foreign to a particular biological system, such as a cell or host cell, and does not naturally exist in that system, and can be introduced into the system by artificial or recombinant means. Thus, the expression of a heterologous TCR or CAR can change the immunological specificity of T cells, so that they recognize or exhibit improved recognition of one or more cancer and / or tumor antigens or peptide antigens thereof present on the surface of cancer cells of individuals with cancer. The modification of T cells and their subsequent expansion can be carried out in vitro and / or ex vivo.

[0017] According to the present invention, the cancer and / or tumor antigen or peptide antigen thereof can be a cancer-testis antigen, NY-ESO-1, MART-1 (a melanoma antigen recognized by T cells), WT1 (Wilms' tumor 1), gp100 (glycoprotein 100), tyrosinase, PRAME (an antigen preferentially expressed in melanoma), p53, HPV-E6 / HPV-E7 (human papillomavirus), HBV, TRAIL, DR4, thyroglobulin, TGFBII frameshift antigen, LAGE-1A, KRAS, CMV (cytomegalovirus), CEA (carcinoembryonic antigen), AFP (alpha-fetoprotein), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, and MAGE-A9, MAGE-A10, or MAGE-A12, or a peptide antigen thereof. Preferably, the tumor antigen is MAGE-A4 or AFP or a peptide antigen thereof. Preferably, the cancer and / or tumor antigen peptide is a peptide antigen of MAGE-A4 and / or comprises the amino acid sequence GVYDGREHTV, SEQ ID NO: 1 (MAGE-A4), or a peptide antigen of alpha-fetoprotein (AFP) and / or comprises the sequence FMNKFIYEI (SEQ ID NO: 2) or residues 158-166 from alpha-fetoprotein (AFP) (SEQ ID NO: 3).

[0018] According to the present invention, the xenogeneic TCR or CAR binds or specifically binds to a cancer and / or tumor antigen or peptide antigen thereof associated with a tumor or cancerous condition, and / or is presented by a tumor or cancer cell or tissue, and / or binds or specifically binds to a tumor cell and / or tissue and / or cancer cell and / or tissue of a subject, patient, or cancer patient suffering from a disease state or cancerous condition. The subject, patient, or cancer patient can then be treated with an engineered T cell or population thereof according to the present invention. Cancer patients suitable for treatment according to the present invention with engineered T cells can be identified by a method comprising the steps of obtaining a sample of tumor and / or cancer cells from an individual or subject with a tumor and / or cancer, and identifying the cancer cells as binding to the expressed xenogeneic TCR or CAR.

[0019] Specificity describes the strength of binding between a heterologous TCR or CAR and a specific target cancer and / or tumor antigen or its peptide antigen, and can be described by the dissociation constant, Kd, ​​i.e., the ratio of the bound state to the unbound state for the receptor-ligand system. In addition, the fewer different cancer and / or tumor antigens or their peptide antigens a heterologous TCR or CAR can bind, the greater its binding specificity. According to the present invention, a heterologous TCR or CAR can bind to less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 different cancer and / or tumor antigens or their peptide antigens.

[0020] According to the invention, the xenogeneic TCR or CAR may be present in a concentration of 0.01 μM to 100 μM, 0.01 μM to 50 μM, 0.01 μM to 20 μM, 0.05 μM to 20 μM or 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM, 5.5 μM, 6.0 μM, 6.5 μM, 7.0 μM, 7.5 μM, 8.0 μM, 8.5 μM, 9.0 μM, 9.5 μM, 10.0 μM; also can bind with a dissociation constant of 10 μM to 1000 μM, 10 μM to 500 μM, 50 μM to 500 μM, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, or 500 μM. Dissociation constant, K D or k off / k on is the dissociation rate constant k off , and the binding rate constant k on The TCR dissociation constant can be determined by experimentally measuring the TCR α chain variable domain and the TCR β chain variable domain.

[0021] Thus, a xenogeneic TCR or CAR for use according to the present invention may optionally be coupled to a peptide-presenting molecule, such as an HLA, e.g., HLA-A * 02 and optionally HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, HLA-A * 02:05, HLA-A *02:06, HLA-A * 02:642 or HLA-A * 02:07, preferably HLA-A * 02:01 or HLA-A * It is capable of binding efficiently and / or with high affinity to cancer and / or tumor antigens or MAGE-A4 or AFP peptide antigens, preferably peptides comprising GVYDGREHTV, SEQ ID NO: 1 (MAGE-A4 peptide) or FMNKFIYEI (AFP peptide) SEQ ID NO: 2, for example, with a dissociation constant of 0.01 μM to 100 μM, for example, 50 μM, 100 μM, 200 μM, 500 μM, preferably 0.05 μM to 20.0 μM, in a complex with 02:642 or without presentation in a complex with a peptide presenting molecule, e.g., HLA. For example, the heterologous TCR or CAR can have binding properties for endogenously expressed tumor cell surface cancer and / or tumor antigens or peptide antigens thereof, optionally wherein the binding is independent of presentation of the cell surface antigen as a complex with a peptide-presenting or antigen-presenting molecule, e.g., major histocompatibility complex (MHC) or human leukocyte antigen (HLA) or major histocompatibility complex class-related protein (MR)1.

[0022] According to the present invention, TCR or CAR binding can be, optionally, specific for one cancer and / or tumor antigen, e.g., a MAGE protein such as MAGE-A4 or AFP, or its peptide antigen, compared to a closely related cancer and / or tumor antigen or peptide antigen sequence. The closely related cancer and / or tumor antigen or peptide antigen sequence can have a similar or identical length and / or a similar or identical number of amino acid residues. The closely related peptide antigen sequence can share 50, 60, 70, or 80-90% identity, preferably 80-90% identity, and / or can differ by 1, 2, 3, or 4 amino acid residues. The closely related peptide sequence can be derived from a sequence containing the polypeptide sequence GVYDGREHTV (SEQ ID NO: 1) or FMNKFIYEI (SEQ ID NO: 2). Binding affinity can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE™ analysis). Avidity is the sum of the strength of binding between two molecules at multiple sites, taking into account, for example, the valency of the interaction. According to the present invention, immunoresponsive cells can exhibit improved affinity and / or avidity for a cancer and / or tumor antigen or peptide antigen thereof, or a cancer and / or tumor antigen or peptide antigen thereof presented by a tumor in a cancer cell or tissue and recognized by a heterologous TCR or CAR, compared to T cells lacking a heterologous TCR or CAR or bearing another heterologous TCR or CAR.

[0023] According to the present invention, heterologous TCR or CAR can optionally selectively bind to cancer and / or tumor antigens or their peptide antigens as described herein above, which are associated with cancerous conditions and / or presented by tumors in cancer cells or tissues; optionally, cancer and / or tumor antigens or their peptide antigens are recognized by heterologous TCR or CAR, optionally in a complex with a peptide-presenting molecule, such as HLA, which is preferably expressed by tumor cells or cancer cells or tissues, or without being presented in a complex with a peptide-presenting molecule or HLA.Selective binding means that heterologous TCR or CAR binds to a cancer and / or tumor antigen or its peptide antigen with greater affinity than another antigen.Selective binding is represented by the equilibrium constant for the displacement of a ligand antigen by another ligand antigen in the complex with heterologous TCR or CAR.

[0024] According to the present invention, the heterologous TCR or CAR binding is selective and / or specific for cancer and / or tumor antigens or peptide antigens as described herein. According to the present invention, the heterologous TCR or CAR can bind and / or specifically bind and / or selectively bind to a peptide-presenting molecule, e.g., HLA, that presents or displays a cancer and / or tumor antigen or a peptide antigen thereof, i.e., a peptide fragment (pHLA) of the cancer and / or tumor antigen, wherein the HLA corresponds to MHC class I (A, B, and C) or specific alleles thereof, all of which are HLA class 1, or the HLA corresponds to MHC class II (DP, DM, DO, DQ, and DR) or specific alleles thereof, preferably the HLA is class 1, and preferably the allele is HLA-A2 or HLA-A3. * 02 or HLA-A2+ or HLA-A * HLA-02 positive, optionally HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, HLA-A *02:05, HLA-A * 02:06, HLA-A * 02:642 or HLA-A * 02:07, preferably HLA-A * 02:01 or HLA-A * 02:642. Alternatively, the xenogeneic TCR or CAR can bind and / or specifically bind and / or selectively bind to a cancer and / or tumor antigen or peptide antigen thereof that is not presented or displayed by a peptide presenting molecule, e.g., HLA.

[0025] Heterogeneous TCR Preferably, the heterologous TCR or CAR is not naturally expressed by the immunoresponsive cell (i.e., the TCR or CAR is exogenous or heterologous). The heterologous TCR may comprise an αβ TCR heterodimer. The heterologous TCR or CAR may be a recombinant, synthetic, or artificial TCR or CAR, i.e., a non-naturally occurring TCR. For example, the heterologous TCR can be genetically engineered to increase its affinity or avidity for a specific cancer and / or tumor antigen or its peptide antigen (i.e., an affinity-enhanced TCR or a specific peptide-enhanced affinity receptor (SPEAR) TCR). The affinity-enhanced TCR or (SPEAR) TCR may contain one or more mutations relative to a naturally occurring TCR, for example, one or more mutations in the hypervariable complementarity-determining regions (CDRs) of the variable regions of the TCR α and β chains. These mutations can optionally increase the affinity of the TCR for MHC displaying a peptide fragment of a tumor antigen when expressed by a tumor and / or cancer cell. Suitable methods for generating affinity-enhanced or matured TCRs include screening libraries of TCR mutants using phage or yeast display and are well known in the art (see, e.g., Robbins et al J Immunol (2008) 180(9):6116; San Miguel et al (2015) Cancer Cell 28 (3) 281-283; Schmitt et al (2013) Blood 122 348-256; Jiang et al (2015) Cancer Discovery 5 901). Preferred enhanced affinity TCRs are capable of binding to tumors or cancer cells expressing a tumor antigen of the MAGE family, such as MAGE-A4 or a peptide antigen thereof, such as a peptide antigen of the sequence GVYDGREHTV, SEQ ID NO: 1 or alpha-fetoprotein (AFP) and / or a tumor antigen comprising the sequence FMNKFIYEI (SEQ ID NO: 2) or residues 158 to 166 from alpha-fetoprotein (AFP) (SEQ ID NO: 3).

[0026] According to the present invention, the heterologous TCR may be a MAGE-A4 TCR which may comprise an alpha chain reference amino acid sequence of SEQ ID NO: 4 or a variant thereof and a beta chain reference amino acid sequence of SEQ ID NO: 6 or a variant thereof.

[0027] According to an alternative embodiment, the heterologous TCR may be an AFP TCR which may comprise an alpha chain reference amino acid sequence of SEQ ID NO: 16 or a variant thereof and a beta chain reference amino acid sequence of SEQ ID NO: 18 or a variant thereof.

[0028] A variant may have an amino acid sequence with at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a reference amino acid sequence.

[0029] The TCR may be encoded by an alpha chain reference nucleotide sequence of SEQ ID NO: 5 or a variant thereof and a beta chain reference nucleotide sequence of SEQ ID NO: 7 or a variant thereof.

[0030] According to an alternative embodiment, the TCR may be encoded by an alpha chain reference nucleotide sequence of SEQ ID NO: 17 or a variant thereof and a beta chain reference nucleotide sequence of SEQ ID NO: 19 or a variant thereof.

[0031] A variant may have a nucleotide sequence with at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a reference nucleotide sequence.

[0032] According to the present invention, the TCR (MAGE-A4 TCR) may comprise a TCR alpha chain variable domain and a TCR beta chain variable domain, (i) the α chain variable domain has the sequence VSPFSN (αCDR1), amino acids 48 to 53 of SEQ ID NO: 10 or SEQ ID NO: 4; LTFSEN(αCDR2), amino acids 71-76 of SEQ ID NO: 11 or SEQ ID NO: 4, and CVVSGGTDSWGKLQF (αCDR3), amino acids 111 to 125 of SEQ ID NO: 12 or SEQ ID NO: 4 and (ii) the β-chain variable domain has the sequence KGHDR (βCDR1), amino acids 46 to 50 of SEQ ID NO: 13 or SEQ ID NO: 6; SFDVKD(βCDR2), amino acids 68 to 73 of SEQ ID NO: 14 or SEQ ID NO: 6, and CATSGQGAYEEQFF (βCDR3), amino acids 110 to 123 of SEQ ID NO: 15 or SEQ ID NO: 6 or These include sequences having at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, optionally 100% sequence identity thereto.

[0033] According to an alternative embodiment, the TCR (AFP TCR) may comprise a TCR alpha chain variable domain and a TCR beta chain variable domain, (i) the α chain variable domain has the sequence DRGSQS (αCDR1), amino acids 27 to 32 of SEQ ID NO: 22 or SEQ ID NO: 16, IYSNGD (αCDR2), amino acids 50 to 55 of SEQ ID NO: 23 or SEQ ID NO: 16, and AVNSDSGYALNF(αCDR3), amino acids 90 to 101 of SEQ ID NO: 24 or SEQ ID NO: 16 and (ii) the β-chain variable domain has the sequence SGDLS (βCDR1), amino acids 27 to 31 of SEQ ID NO: 25 or SEQ ID NO: 18, YYNGEE (βCDR2), amino acids 49 to 54 of SEQ ID NO: 26 or SEQ ID NO: 18, and ASSLGGESEQY (βCDR3), amino acids 92 to 102 of SEQ ID NO: 27 or SEQ ID NO: 18 or These include sequences having at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, optionally 100% sequence identity thereto.

[0034] According to an alternative embodiment, the TCR may be substituted: a. αCDR1 having the sequence DRGSQA, SEQ ID NO: 28; b. αCDR2 having the sequence AVNSDSSYALNF, SEQ ID NO: 29; c. αCDR2 having the sequence AVNSDSGVALNF, SEQ ID NO: 30; d. αCDR1 having the sequence DRGSQA, SEQ ID NO:28 and αCDR2 having the sequence AVNSDSGVALNF, SEQ ID NO:30; e. αCDR2 having the sequence AVNSQSGYALNF, SEQ ID NO: 31; f. αCDR2 having the sequence AVNSQSGYSLNF, SEQ ID NO: 32; g. αCDR2 having the sequence AVNSQSSYALNF, SEQ ID NO: 36; h. an αCDR1 having the sequence DRGSQA, SEQ ID NO: 28, and an αCDR2 having the sequence AVNSQSGYALNF, SEQ ID NO: 31; i. αCDR2 having the sequence AVNSQSGVALNF, SEQ ID NO: 32; j. αCDR2 having the sequence AVNSQNGYALNF, SEQ ID NO: 33; k. αCDR1 having the sequence DRGSFS, SEQ ID NO: 34; l. αCDR1 having the sequence DRGSYS, SEQ ID NO: 35; m. αCDR1 having the sequence DRGSYS, SEQ ID NO: 35 and αCDR2 having the sequence AVNSDSSYALNF, SEQ ID NO: 29; n. αCDR1 having the sequence DRGSYS, SEQ ID NO: 35 and αCDR2 having the sequence AVNSDSSYALNF, SEQ ID NO: 29; o. αCDR1 having the sequence DRGSYS, SEQ ID NO:35 and αCDR2 having the sequence AVNSQSGYALNF, SEQ ID NO:31.

[0035] Thus, a TCR (MAGE-A4 TCR) may comprise a TCR in which the alpha chain variable domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of amino acid residues 1 to 136 of SEQ ID NO: 8 or SEQ ID NO: 4, and / or the beta chain variable domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of amino acid residues 1 to 133 of SEQ ID NO: 9 or SEQ ID NO: 6.

[0036] Alternatively, the TCR (AFP TCR) may comprise a TCR wherein the alpha chain variable domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of amino acid residues 1 to 112 of SEQ ID NO: 20 or SEQ ID NO: 16, and / or the beta chain variable domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of amino acid residues 1 to 112 of SEQ ID NO: 21 or SEQ ID NO: 18.

[0037] The terms "precursor TCR" or "parent TCR" are used herein to refer to a TCR comprising the MAGE-A4 TCR α and MAGE-A4 TCR β chains of SEQ ID NOS: 4 and 6, respectively, or the AFP TCR α and AFP TCR β chains of SEQ ID NOS: 16 and 18, respectively. It is desirable to provide TCRs that are mutated or modified relative to the precursor TCR, which have the same, equivalent, or higher affinity and / or the same, equivalent, or slower off-rate for peptide-HLA complexes than the precursor TCR. According to the present invention, the heterologous TCR may have two or more mutations in the α and / or β chain variable domains relative to the precursor TCR, and may be referred to as "engineered TCRs" or "mutant TCRs." These mutations may improve binding affinity and / or specificity and / or selectivity and / or avidity for MAGE-A4 or its peptide antigen. In particular embodiments, there are 1, 2, 3, 4, 5, 6, 7 or 8 mutations in the alpha chain variable domain, for example 4 or 8 mutations, and / or 1, 2, 3, 4 or 5 mutations in the beta chain variable domain, for example 5 mutations. In some embodiments, the alpha chain variable domain of a TCR of the invention may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence of amino acid residues of SEQ ID NO: 8 for MAGE-A4 TCR and SEQ ID NO: 20 for AFP TCR. In some embodiments, the beta chain variable domain of a TCR of the present invention may comprise an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence of amino acid residues of SEQ ID NO: 9 for MAGE-A4 TCR or SEQ ID NO: 21 for AFP TCR.

[0038] According to the present invention, a TCR is provided in which the α chain variable domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of amino acid residues 1 to 136 of SEQ ID NO: 8 or SEQ ID NO: 4, or the amino acid residues 1 to 47, 54 to 70, 77 to 110 and 126 to 136 thereof are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of amino acid residues 1 to 47, 54 to 70, 77 to 110 and 126 to 136 of SEQ ID NO: 8, respectively. The TCR may comprise a TCR (MAGE-A4 TCR) having a sequence identity with the amino acid residues 48 to 53, 71 to 76, and 111 to 125 of SEQ ID NO: 8, respectively, and / or wherein amino acid residues 48 to 53, 71 to 76, and 111 to 125 of SEQ ID NO: 8, respectively, CDR1, CDR2, and CDR3 have an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acid residues 48 to 53, 71 to 76, and 111 to 125 of SEQ ID NO: 8, respectively, CDR1, CDR2, and CDR3.

[0039] According to the present invention, TCR (MAGE-A4) comprises, in the α chain variable domain: (i) its amino acid residues 1-47 may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 1-47 of SEQ ID NO: 8, or (b) have 1, 2 or 3 amino acid residues inserted or deleted relative to residues 1-47 of SEQ ID NO: 8; (ii) amino acid residues 48 to 53 are amino acids 48 to 53 of VSPFSN, CDR1, SEQ ID NO: 10, or SEQ ID NO: 8; (iii) amino acid residues 54-70 thereof may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 54-70 of SEQ ID NO: 25, or (b) have one, two or three amino acid residues inserted or deleted relative to the sequence of amino acid residues 54-70 of SEQ ID NO: 8; (iv) amino acid residues 71-76 can be amino acids 71-76 of LTFSEN, CDR2, SEQ ID NO:11, or SEQ ID NO:8; (v) amino acid residues 77-110 thereof may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 77-110 of SEQ ID NO: 8, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 77-110 of SEQ ID NO: 8; (vi) amino acids 111-125 can be CVVSGGTDSWGKLQF, CDR3, amino acids 111-125 of SEQ ID NO: 12, or SEQ ID NO: 8; (vii) the amino acid residues 126 to 136 may have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 126 to 136 of SEQ ID NO: 8, or may have one, two, or three insertions, deletions, or substitutions relative to the sequence of amino acid residues 126 to 136 of SEQ ID NO: 8; The TCR may comprise a TCR of the sequence:

[0040] According to the present invention, the TCR may comprise a TCR whose β chain variable domain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which amino acid residues 1 to 45, 51 to 67, 74 to 109, and 124 to 133 have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequences of amino acid residues 1 to 45, 51 to 67, 74 to 109, and 124 to 133 of SEQ ID NO: 9, respectively, and amino acid residues 46 to 50, 68 to 73, and 110 to 123 have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequences of amino acid residues 46 to 50, 68 to 73, and 110 to 123 of SEQ ID NO: 9, CDR1, CDR2, and CDR3, respectively, or a MAGE-A4 TCR.

[0041] According to the present invention, the TCR comprises, in the β chain variable domain: (i) its amino acid residues 1-45 can have (a) at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 1-45 of SEQ ID NO: 9, or (b) 1, 2 or 3 amino acid residues inserted or deleted relative to residues 1-45 of SEQ ID NO: 9; (ii) amino acid residues 46 to 50 are amino acids 46 to 50 of KGHDR, CDR1, SEQ ID NO: 13, or SEQ ID NO: 9; (iii) its amino acid residues 51-67 may (a) have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 51-67 of SEQ ID NO: 9, or (b) have one, two, or three amino acid residues inserted or deleted relative to the sequence of amino acid residues 51-67 of SEQ ID NO: 9; (iv) amino acid residues 68-73 can be amino acids 68-73 of SFDVKD, CDR2, SEQ ID NO: 14, or SEQ ID NO: 9; (v) amino acid residues 74 to 109 may have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 74 to 109 of SEQ ID NO: 9, or may have one, two, or three insertions, deletions, or substitutions relative to the sequence of amino acid residues 74 to 109 of SEQ ID NO: 9; (vi) amino acids 110-123 can be CATSGQGAYEEQFF, CDR3, amino acids 110-123 of SEQ ID NO: 15, or SEQ ID NO: 9; (vii) the amino acid residues 124 to 133 thereof may have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 124 to 133 of SEQ ID NO: 9, or may have one, two, or three insertions, deletions, or substitutions relative to the sequence of amino acid residues 124 to 133 of SEQ ID NO: 9; The TCR may comprise a TCR of the sequence:

[0042] Alternatively, the TCR may comprise an α chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of amino acid residues 1 to 112 of SEQ ID NO: 16, or to the sequences of amino acid residues 1 to 26, 33 to 49, 56 to 89 and 102 to 112 of SEQ ID NO: 16, respectively. and / or the amino acid residues 27 to 32, 50 to 55, 90 to 101, respectively, of CDR1, CDR2, and CDR3 of SEQ ID NO: 16 have an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences of amino acid residues 27 to 32, 50 to 55, 90 to 101, respectively, of CDR1, CDR2, and CDR3 of SEQ ID NO: 16, or an AFP TCR.

[0043] According to the present invention, the TCR comprises in the alpha chain variable domain: (i) its amino acid residues 1-26 can have (a) at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 1-26 of SEQ ID NO: 16, or (b) one, two or three amino acid residues inserted or deleted relative to residues 1-26 of SEQ ID NO: 16; (ii) amino acid residues 27 to 32 are DRGSQS , αCDR1, amino acids 27 to 32 of SEQ ID NO: 22 or SEQ ID NO: 16; (iii) its amino acid residues 33-49 may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 33-49 of SEQ ID NO: 16, or (b) have one, two or three amino acid residues inserted or deleted relative to the sequence of amino acid residues 33-49 of SEQ ID NO: 16; (iv) amino acid residues 50 to 55 are IYSNGD , αCDR2, amino acids 50-55 of SEQ ID NO: 23 or SEQ ID NO: 16; (v) the amino acid residues 56 to 89 may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 56 to 89 of SEQ ID NO: 16, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 56 to 89 of SEQ ID NO: 16; (vi) amino acids 90 to 101 are AVNSDSGYALNF , αCDR3, amino acids 90-101 of SEQ ID NO: 24 or SEQ ID NO: 16; (vii) the amino acid residues 102 to 112 may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 102 to 112 of SEQ ID NO: 16, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 102 to 112 of SEQ ID NO: 16; The TCR may comprise a TCR of the sequence:

[0044] According to the present invention, a TCR or AFP TCR may comprise a TCR whose β chain variable domain comprises the amino acid sequence of amino acid residues 1 to 112 of SEQ ID NO: 18, or an amino acid sequence in which amino acid residues 1 to 26, 32 to 48, 55 to 91, and 103 to 112 thereof have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequences of amino acid residues 1 to 26, 32 to 48, 55 to 91, and 103 to 112 of SEQ ID NO: 18, respectively, and amino acid residues 27 to 31, 49 to 54, and 92 to 102 have at least 70%, 75%, 80%, 85%, 90% or 95% identity to amino acid residues 27 to 31, 49 to 54, and 92 to 102 of SEQ ID NO: 18, the sequences of βCDR1, βCDR2, and βCDR3, respectively.

[0045] According to the present invention, the TCR comprises, in the β chain variable domain: (i) its amino acid residues 1-26 may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 1-26 of SEQ ID NO: 18, or (b) have 1, 2 or 3 amino acid residues inserted or deleted relative to residues 1-26 of SEQ ID NO: 18; (ii) amino acid residues 27 to 31 are SGDLS βCDR1, amino acids 27 to 31 of SEQ ID NO: 25 or SEQ ID NO: 18; (iii) its amino acid residues 32 to 48 may (a) have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 32 to 48 of SEQ ID NO: 18, or (b) have one, two, or three amino acid residues inserted or deleted relative to the sequence of amino acid residues 32 to 48 of SEQ ID NO: 18; (iv) amino acid residues 49 to 54 are YYNGEE βCDR2, amino acids 49-54 of SEQ ID NO: 26 or SEQ ID NO: 18; (v) amino acid residues 55-91 thereof may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 55-91 of SEQ ID NO: 18, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 55-91 of SEQ ID NO: 18; (vi) amino acids 92 to 102 are ASSLGGESEQY βCDR3, amino acids 92-102 of SEQ ID NO: 27 or SEQ ID NO: 18; (vii) the amino acid residues 103 to 112 may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 103 to 112 of SEQ ID NO: 18, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 103 to 112 of SEQ ID NO: 18; The TCR may comprise a TCR of the sequence:

[0046] Thus, a heterologous TCR may comprise a TCR whose alpha chain comprises amino acid residues of SEQ ID NO:20 and whose beta chain variable domain comprises amino acid residues of SEQ ID NO:21 or SEQ ID NO:42.

[0047] Coreceptor-modified T cells According to the present invention, a population of engineered T cells expressing or displaying a heterologous TCR or CAR can further express or display a heterologous co-receptor. The heterologous co-receptor can be a CD8 co-receptor. The CD8 co-receptor can comprise a dimer or pair of CD8 chains, including CD8-α and CD8-β chains or CD8-α and CD8-α chains. Preferably, the CD8 co-receptor is a CD8αα co-receptor, including CD8-α and CD8-α chains. The CD8α co-receptor can comprise an amino acid sequence at least 80% identical to SEQ ID NO: 37, SEQ ID NO: 37, or a variant thereof. The CD8α co-receptor can be a homodimer.

[0048] The CD8 co-receptor binds to class 1 MHC and enhances TCR signaling. According to the present invention, the CD8 co-receptor may have the reference amino acid sequence of SEQ ID NO: 37 or may be a variant thereof. The variant may have an amino acid sequence having at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the reference amino acid sequence of SEQ ID NO: 37. The CD8 co-receptor may be encoded by the reference nucleotide sequence of SEQ ID NO: 38 or may be a variant thereof. Variants may have a nucleotide sequence with at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the reference nucleotide sequence SEQ ID NO:38.

[0049] According to the present invention, the heterologous CD8 co-receptor comprises, in the Ig-like V-type domain, the sequence: (i) VLLSNPTSG, CDR1, SEQ ID NO: 39, or amino acids 45 to 53 of SEQ ID NO: 37; (ii) YLSQNKPK, CDR2, amino acids 72 to 79 of SEQ ID NO: 40 or SEQ ID NO: 37; (iii) LSNSIM, CDR3, amino acids 80 to 117 of SEQ ID NO: 41 or SEQ ID NO: 37; or a sequence having at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. The CD8 co-receptor may comprise a CD8 co-receptor comprising a CDR having the following sequence:

[0050] According to the present invention, a heterologous CD8 co-receptor may comprise a CD8 co-receptor comprising, or in an Ig-like V-domain, residues 22 to 135 of the amino acid sequence of SEQ ID NO: 37, or amino acid residues 22 to 44, 54 to 71, 80 to 117, 124 to 135 thereof, which have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the amino acid residues 22 to 44, 54 to 71, 80 to 117, 124 to 135 of SEQ ID NO: 37, CDR1, CDR2, CDR3, respectively, and amino acid residues 45 to 53, 72 to 79, and 118 to 123 thereof, which have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the amino acid residues 45 to 53, 72 to 79, and 118 to 123 of SEQ ID NO: 37, respectively.

[0051] According to the present invention, the CD8 co-receptor is (i) its amino acid residues 22-44 may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 22-44 of SEQ ID NO: 37, or (b) have 1, 2 or 3 amino acid residues inserted or deleted relative to residues 22-44 of SEQ ID NO: 37; (ii) amino acid residues 45 to 53 are amino acids 45 to 53 of VLLSNPTSG, SEQ ID NO: 39, CDR1, or SEQ ID NO: 37; (iii) amino acid residues 54-71 thereof may (a) have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 54-71 of SEQ ID NO: 37, or (b) have one, two or three amino acid residues inserted or deleted relative to the sequence of amino acid residues 54-71 of SEQ ID NO: 37; (iv) amino acid residues 72-79 can be amino acids 72-79 of YLSQNKPK, CDR2, SEQ ID NO: 40, or SEQ ID NO: 37; (v) the amino acid residues 80 to 117 may have at least 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence of amino acid residues 80 to 117 of SEQ ID NO: 37, or may have one, two or three insertions, deletions or substitutions relative to the sequence of amino acid residues 80 to 117 of SEQ ID NO: 37; (vi) amino acids 118-123 can be amino acids 80-117 of LSNSIM, CDR3, SEQ ID NO:41, or SEQ ID NO:37; (vii) amino acid residues 124 to 135 thereof may have at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequence of amino acid residues 124 to 135 of SEQ ID NO: 37, or may have one, two, or three insertions, deletions, or substitutions relative to the sequence of amino acid residues 124 to 135 of SEQ ID NO: 37; or in the Ig-like V-type domain, the sequence of the CD8 co-receptor.

[0052] Engineered T cells expressing a heterologous CD8 co-receptor can exhibit improved affinity and / or avidity and / or improved T cell activation, as determinable by the assays disclosed herein, to or upon stimulation with an antigenic peptide, tumor or cancer antigen, optionally when presented on HLA, compared to engineered T cells that do not express the heterologous CD8 co-receptor. The heterologous CD8 of the engineered T cells can interact with or specifically bind to MHC, which can be a class I or class II, preferably class I major histocompatibility complex (MHC), an HLA-I molecule, or can involve an MHC class I HLA-A / B2M dimer, and preferably CD8-α interacts with the α3 portion (residues 223-229) of class I MHC, preferably via the IgV-like domain of CD8. Thus, the xenogeneic CD8 improves TCR binding of the T cell to HLA and / or antigenic peptides that bind to or are presented by HLA pMHCI or pHLA, optionally on the surface of antigen-presenting cells, dendritic cells and / or tumor or cancer cells, tumor or cancer tissue, compared to T cells lacking the xenogeneic CD8.

[0053] Thus, xenogeneic CD8 reduces the off-rate (k) of cell (TCR) / peptide-major histocompatibility complex class I (pMHCI) interactions of immunoresponsive cells, optionally on the surface of antigen-presenting cells, dendritic cells, and / or tumor or cancer cells, or tumor or cancer tissue, compared to cells lacking xenogeneic CD8. off), and therefore, its half-life, can be improved or increased, thereby also providing improved ligation affinity and / or avidity. Heterologous CD8 can improve TCR organization on the surface of immunoresponsive cells, thereby enabling cooperation in pHLA binding and providing improved therapeutic avidity. Thus, heterologous CD8 co-receptor-modified T cells can bind or interact with LCK (lymphocyte-specific protein tyrosine kinase) in a zinc-dependent manner, resulting in the activation of transcription factors such as NFAT, NF-κB, and AP-1. Heterologous CD8-modified T cells can have improved or increased CD40L expression, cytokine production, cytotoxic activity, induction of dendritic cell maturation, or induction of dendritic cell cytokine production, optionally in response to cancer and / or tumor antigens or peptide antigens thereof, when presented by cancer cells or tumors in tissues, compared to T cells lacking the heterologous CD8 co-receptor.

[0054] Co-stimulatory ligand-modified T cells According to the present invention, the engineered T cells or populations of engineered T cells can further comprise and / or express at least one, and optionally one, two, three, or four, exogenous and / or recombinant costimulatory ligands. The interaction between the TCR and at least one exogenous costimulatory ligand can result in a non-antigen-specific signal and cell activation. Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share many common characteristics. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor β (TNFP) / lymphotoxin-α (LTa), lymphotoxin-β (TTb), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess immunoglobulin domains (folds). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both of which are ligands for CD28.In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD275, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof. In accordance with the present invention, the engineered T cells or populations of engineered T cells may further comprise at least one exogenous and / or recombinant costimulatory ligand, which may be 4-1BBL or CD80, preferably 4-1BBL, alternatively 4-1BBL and CD80.

[0055] CD3+ enrichment According to the present invention, the method may further comprise a step in which T cells are enriched for T cells expressing antigens, cluster of differentiation proteins, and CD3, which is part of the T cell receptor (TCR) complex on mature T lymphocytes, with respect to the CD3+ fraction. Enrichment can be performed before the modification of T cells. Enrichment can be performed before the activation of T cells. Alternatively, and preferably, enrichment is performed during or after the activation of T cells, preferably either during or after the activation of T cells and before the modification. Optionally, enrichment is performed on the included T cells against an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof, optionally attached to removable beads, for example, during activation, optionally before the modification.

[0056] T cell activation According to the methods of the present invention, the activation or activation step of a T cell or population of T cells stimulates the T cells to proliferate and / or expand.

[0057] Activation of an isolated population of T cells can be achieved by various methods, for example, by contacting the T cells with an anti-CD3 antibody or a CD3-binding fragment thereof, or by contacting the T cells with an anti-CD28 antibody or a CD28-binding fragment thereof, or by contacting the T cells with B7 protein (B7 is a type of peripheral membrane protein found on activated antigen-presenting cells that can generate a costimulatory signal when paired with CD28 or CD152 (CTLA-4) surface protein on T cells) or a CD28-binding fragment thereof, e.g., B7-1 or B7-2 or a CD28-binding fragment thereof. The activation means can be attached to a solid and optionally releasable surface or substrate such as beads or magnetic beads, for example, magnetic beads coated with anti-CD3 and / or anti-CD28. Preferably, activation is by the addition of an anti-CD3 antibody or antigen-binding fragment thereof and / or an anti-CD28 antibody or antigen-binding fragment thereof, optionally attached to beads, which may be, for example, magnetic beads and thus can be isolated from the cell culture medium, optionally removable beads. T cell activation can be performed simultaneously with or after T cell modification, simultaneously with or after AKTi addition, or simultaneously with or after both T cell modification and AKTi addition. Preferably, T cell activation precedes T cell modification, preferably precedes AKTi addition, preferably precedes both T cell modification and AKTi addition.

[0058] AKT inhibitor steps According to the methods of the invention, T cell modification or transduction can occur prior to or concurrently with activation, for example, at any one of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours or more before activation.

[0059] According to the methods of the invention, T cell modification or transduction can be performed after activation, preferably 18-26 hours after activation, preferably any one of 12-40, 13-38, 14-36, 15-34, 16-32, 17-30, 18-28, 18-26 or 18-24 hours after activation or any one of about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 hours after activation.

[0060] According to the methods of the present invention, the AKT inhibitor can be added after T cell modification or transduction, preferably the AKT inhibitor can be added any one of 8 to 42, 9 to 41, 10 to 40, 11 to 39, 12 to 38, 13 to 37, 14 to 36, 15 to 35, 16 to 34, 17 to 33, 18 to 32, 19 to 31, 20 to 30, 21 to 29, 22 to 28, 23 to 27, 24 to 26 or 24 to 25 hours after modification or transduction, preferably any one of 15 to 26, 16 to 25, 17 to 24, 18 to 23, 19 to 22 or 20 to 21 hours after T cell modification or transduction, preferably 17 to 24 hours.

[0061] According to the methods of the present invention, the AKT inhibitor can be added after T cell activation, preferably, the AKT inhibitor can be added at any one of 35 to 50, 36 to 49, 37 to 48, 38 to 47, 39 to 46, 40 to 45, 41 to 44, and 42 to 43 hours after T cell activation, preferably 35 to 50 hours after T cell modification or transduction.

[0062] AKT inhibitors According to the method of the present invention, the AKT inhibitor can be selected from the group consisting of an allosteric inhibitor or allosteric AKT inhibitor, a competitive ATP inhibitor, an inhibitor of the interaction between AKT and phospholipids, an inhibitor of the phosphorylation of a molecule downstream of AKT, preferably PRAS40, ribosomal S6, or TSC2. The AKT inhibitor can also be selected from the group consisting of an inhibitor of DNA-PK activation of AKT, an inhibitor of PDK-1 activation of AKT, an inhibitor of mTORC2 activation of AKT, and an inhibitor of HSP activation of AKT.

[0063] Therefore, allosteric inhibitors or allosteric AKT inhibitors: (a) ARQ092, miransertib, CAS number: 1313881-70-7, formula C 27 H 24 N6, or structure

[0064] [ka] A compound having (b) ARQ751, or structure

[0065] [ka] A compound having (c)BAY1125976, (CAS number 1402608-02-9), formula C 23 H 21 N5O or structure

[0066] [ka] or a compound having (d)MK-2206, (CAS number 1032350-13-2), formula C 25 H 21 N5O or structure

[0067] [ka] A compound having You can choose from one of the following.

[0068] Therefore, competitive ATP inhibitors are (a) Afuresertib (GSK2110183), (CAS number: 1047645-82-8), formula C 18 H 17 Cl2FN4OS·HCl, or structure

[0069] [ka] A compound having (b) Uprosertib, GSK2141795, (CAS number: 1047634-65-0), formula C 18 H 16 Cl2F2N4O2, or structure

[0070] [ka] A compound having (c) GSK690693, (CAS number: 937174-76-0), formula C 21 H 27 N7O3, or structure

[0071] [ka] A compound having (d) Ipatasertib (GDC-0068), (CAS number: 1001264-89-6), formula C 24 H 32 ClN5O2, or structure

[0072] [ka] A compound having (e) LY2780301, or structure

[0073] [ka] A compound having (f) Triciribine (TCN-PM; VD-0002), (CAS number: 35943-35-2), formula C 13 H 16 N6O4, or structure

[0074] [ka] A compound having (g)AZD5363, (CAS1143532-39-1), Formula C 21 H 25 ClN6O2, or structure

[0075] [ka] or a compound having (g)CCT128930, (CAS885499-61-6), Formula C 18 H 20 ClN5, or structure

[0076] [ka] A compound having You can choose from one of the following.

[0077] Therefore, inhibitors of the interaction between AKT and phospholipids include perifosine (D-21266, KRX0401), (CAS157716-52-4), and compounds of formula C 25 H 52 NO4P, or structure

[0078] [ka] The compound may have the formula:

[0079] Preferably, the AKT inhibitor is MK-2206 or GSK690693.

[0080] According to the present invention, the AKT inhibitor can be added at a concentration that is 10 to 1000 times or more greater than the IC50 of the AKT inhibitor for inhibition of AKT, optionally the IC50 for inhibition of AKT1, AKT2, or AKT3, preferably at a concentration that is 10, 25, 20, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times or more greater than the IC50. Preferably, the AKT inhibitor can be added at a concentration of 0.01 μM to 10 μM or 20 μM to 100 μM, preferably 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μM, preferably 0.5 μM or more.

[0081] Process T cells According to the present invention, the activated and modified and / or transduced T cells or populations of T cells produced in the presence of an AKT inhibitor are (i) a population of T cells generated in the absence of an AKT inhibitor, or a population of activated and modified or transduced T cells (e.g., a reference T cell or population of T cells); or (ii) a population of T cells generated in the presence of an AKT inhibitor, or a population of activated and modified or transduced T cells, compared to a population in which the AKT inhibitor is added prior to modification or transduction and / or 24 hours or less after activation or stimulation, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or less after activation or stimulation; (a) T cells expressing both CD45RA+ and CCR7+; (b) T cells that are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+; (c)T SCM T cells, which are stem memory T cells; (d) T cells having a memory phenotype, preferably a CD8 memory phenotype may have an increased or high relative proportion of any one or more of:

[0082] According to the present invention, the activated and modified and / or transduced T cells or populations of T cells produced in the presence of an AKT inhibitor preferably comprise: (i) a population of T cells generated in the absence of an AKT inhibitor, or a population of activated and modified or transduced T cells (e.g., a reference T cell or population of T cells); or (ii) a population of T cells or a population of activated and modified or transduced T cells generated in the presence of an AKT inhibitor, compared to a population in which the AKT inhibitor is added prior to transduction and / or 24 hours or less after activation or stimulation, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or less after activation or stimulation; (a) persistence in vitro and / or in vivo; (b) expansion from seeding to harvesting; (c) durability of response; (d) antigen-induced cytokine production, optionally interferon gamma production (e) T cell survival rate or lifespan or viability percentage; (f) T cell effector function, preferably cytotoxicity have improved levels of any one or more of:

[0083] T cell persistence Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence and has an increased proportion of less terminally differentiated T cells, preferably an increased proportion of double positive SCM cells expressing both CD45RA+ and CCR7+, preferably within the functional CD8+ population. Preferably, the T cells have improved persistence and memory formation, survival, and / or antigen-stimulated survival when tested in vivo and / or in vitro.

[0084] Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence demonstrated in vivo and / or in vitro over a period of time, optionally improved compared to a reference T cell or population of T cells.

[0085] Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence as determined by measuring an increased level of in vivo expansion and / or an increased proportion of double-positive SCM cells, for example, as determined by flow cytometry to identify the proportion of T cells or T cells that express a heterologous TCR or CAR and / or express both CD45RA+, CCR7, optionally compared to a reference T cell or population of T cells, quantified and / or by qPCR to identify genetically modified T cells.

[0086] Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved persistence, as determined, for example, by improved peak or median expansion as a measure of copies / μg of the heterologous TCR or CAR by qPCR, or by measuring, for example, the peak or median percentage of CD3+ cells positive for the heterologous TCR or CAR in a sample of in vivo peripheral blood mononuclear cells (PBMCs). Preferably, these values ​​are improved by at least 10%, alternatively 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or more, compared to a reference T cell or population of T cells. The extent of expansion and duration of persistence correlate with the tumorigenic response.

[0087] Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence as determined by improved levels of cytokine production, e.g., interferon gamma production in vivo and / or in vitro, optionally compared to a reference T cell or population of T cells, as quantified by a cytokine assay, e.g., ELISA, or as described herein.

[0088] Preferably, the activated and modified or transduced T cell or population of T cells undergoes less and / or demonstrates reduced functional attrition in vitro and / or in vivo, optionally improved compared to a reference T cell or population of T cells, e.g., as determined by the assays provided herein, and thus exhibits improved persistence as determined by being relatively able to persist with a longer survival period in vitro and / or in vivo and to provide a longer-lasting and more durable immune response. Preferably, T cell function is enhanced by at least 10%, alternatively 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or more compared to a reference T cell or population of T cells, e.g., as measured by increased secretion of gamma interferon from CD8+ T cells, increased T cell proliferation, e.g., according to cell counting, increased internal signaling, e.g., according to a cell signaling assay, increased antigen responsiveness, increased secretion of cytokines and / or interferons, increased target cell killing, increased T cell activation, increased CD28 signaling, increased ability of T cells to infiltrate tumors, increased ability to recognize and bind to dendritic cell-presented antigens.

[0089] Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence as determined by having improved anti-tumor activity potency, and thus improved reduction of tumor immunity or evasion of immune recognition, as measured, for example, by assays of, or determination of the extent of, tumor infiltration, tumor binding, tumor shrinkage, and / or tumor clearance in vitro and / or in vivo, optionally determined in comparison to a reference T cell or population of T cells. Preferably, the potency of anti-tumor activity is enhanced or improved by at least 10%, alternatively 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or more, as measured, for example, by tumor binding assays, tumor shrinkage, and / or tumor clearance, compared to the reference T cell or population of T cells.

[0090] Preferably, the activated and modified or transduced T cell or population of T cells exhibits improved persistence as determined by, e.g., increased secretion of cytokines and / or interferons, increased T cell proliferation, improved or enhanced tumor immunogenicity as measured by, e.g., at least 5% or 10%, alternatively 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or more, in vitro and / or in vivo, enhanced ability to mount an immune response in response to a tumor or tumor antigen, as measured by, e.g., increased secretion of cytokines and / or interferons, increased T cell proliferation, increased antigen responsiveness, e.g., by in vitro assays, target cell killing, T cell activation, CD28 signaling, ability of T cells to infiltrate tumors, ability to recognize and bind to dendritic cell-presented antigens, compared to a reference T cell or population of T cells.

[0091] Preferably, the activated and modified or transduced T cell or population of T cells exhibits a significant improvement in the survival rate of the T cell or population of T cells, preferably compared to a reference T cell or population of T cells, for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 days, and / or 1, 2, 3, 4, ... The cells exhibit improved persistence over any one or more of the following periods, periods, or time courses: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 months. Preferably, the cells exhibit improved levels of functional activity without attrition over the compared time courses. Preferably, the improved persistence is determined over a period of 8 to 14 days after in vivo injection.

[0092] T cell expansion Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved and / or increased levels of T cell expansion when measured during the process or culture between the time points of seeding and harvest. Preferably, compared to a population of T cells or a population of activated and modified or transduced T cells produced in the absence of an AKT inhibitor (e.g., a reference T cell or population of T cells), e.g., after T cell activation, there is an increased level of T cell expansion, division, or proliferation compared to the reference T cell or population of T cells. For example, assays or determinations of expansion, division, or proliferation can be performed using an automated cell counter that provides measurements of cell viability and concentration and rates of proliferation and expansion measured during or in samples derived from the process or culture process or processes of the invention. Preferably, the activated and modified or transduced T cells or population of T cells exhibit an improved ability to expand, divide, or proliferate compared to a reference T cell or population of T cells when measured in an equivalent sample or sample obtained during the process or culturing process or process of the invention or a sample time point, or a sample derived therefrom, and the improvement can be determined in an assay involving T cell activation. Activation can be initiated in the presence of a cytokine, interleukin, antibody, peptide, or antigenic peptide, as described hereinabove; for example, activation can be via the use of a cancer or tumor antigen or peptide thereof, a peptide fragment of a cancer or tumor antigen recognized by a heterologous TCR, or a cell or tissue, e.g., a tumor or cancer cell or tissue presenting the peptide, antigenic peptide, or peptide fragment. Preferably, the improvement or equivalent improvement compared to the reference is demonstrated over a period or time course, as described hereinabove.

[0093] T cell persistence Thus, the activated and modified or transduced T cells or population of T cells exhibit improved durable responses and / or durable response rates in vivo compared to a reference T cell or population of T cells. Preferably, the T cells or population of T cells provide an improved durable response, e.g., reducing tumor growth or tumor growth rate or maintaining tumor size after cessation of treatment / infusion in vivo, as determined by measuring tumor size or tumor number, preferably enhanced by at least 10%, alternatively 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or more, compared to such levels before infusion or treatment or intervention, or compared to infusion or treatment with a reference T cell or population of T cells. Preferably, the improvement or comparable improvement compared to the reference is demonstrated over a period or time course, as described herein above.

[0094] T cell cytokine production Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved and / or increased levels of cytokine production, e.g., compared to a reference T cell or population of T cells, in response to a cancer or tumor antigen or peptide thereof, or a cancer or tumor antigen peptide, antigenic peptide, or peptide fragment of a cancer or tumor antigen of or presented by a cancer cell or tissue tumor; and recognized by a heterologous TCR or CAR. The cytokine may be granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, IL-2, tumor necrosis factor (TNF)-α, MIP-1β (CCL4), IL-17, IL-10, IL-4, IL-5, IL-13, IL-2 receptor, IL-12, or MIG (CXCL9); preferably IFNγ, IL-2, TNFα, GM-CSF, or MIP1β; preferably IFNγ, IL-2, TNFα, GM-CSF, and MIP1β. Additionally or alternatively, the activated and modified or transduced T cells or population of T cells can exhibit increased levels of cytokine production induction in dendritic cells in response to cancer or tumor antigens or peptides thereof, or peptides, antigenic peptides, or peptide fragments of cancer or tumor antigens of or presented by cancer cells or tissue tumors; and recognized by heterologous TCRs or CARs, compared to a reference T cell or population of T cells. The cytokines can be granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, IL-2, tumor necrosis factor (TNF)-α, MIP-1β (CCL4), IL-17, IL-10, IL-4, IL-5, IL-13, IL-2 receptor, IL-12, or MIG (CXCL9); preferably IFN-γ, IL-12, or MIG; or IFN-γ, IL-12, and MIG. Suitable assays for determining cytokine production are known in the art. Preferably, the improvement or comparable improvement compared to the reference is demonstrated over a period or time course, as described herein above.

[0095] T cell viability, lifespan, viability Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved and / or increased survival rate or lifespan or percentage viability in vitro and / or in vivo, e.g., compared to a reference T cell or population of T cells. Preferably, the improvement or comparable improvement compared to the reference is demonstrated over a period or time course, as described herein above. Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved and / or increased survival rate or lifespan or percentage viability, e.g., compared to a reference T cell or population of T cells, e.g., compared to a reference T cell or population of T cells. Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved and / or increased survival rate or lifespan or percentage viability, e.g., compared to a reference T cell or population of T cells. Preferably, the improved or comparable improvement compared to the reference is demonstrated over a period or time course, as described herein above. 3 The activated and modified or transduced T cells or population of T cells exhibit improved survival, as determined by measuring the level of T cell proliferation in response to antigenic stimulation, e.g., an antigen specific for the heterologous TCR or CAR, in in vitro or in vivo samples using a H-thymidine incorporation proliferation assay. Additionally or alternatively, T cell survival can be determined by measuring the level of antigen-specific response of T cells, e.g., using enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISpot), in terms of cytokine production of antigen-responsive T cells. This can be combined with colorimetric assays for physical cell viability and measurement of marker activities related to the number of viable T cells. Preferably, the activated and modified or transduced T cells or population of T cells exhibit improved persistence, as determined by measuring increased survival or lifespan, e.g., as quantified by flow cytometry to identify the proportion of T cells or T cells expressing a heterologous TCR or CAR and / or expressing both CD45RA+ and CCR7, and / or as determined by qPCR to identify genetically modified T cells.

[0096] T cell effector function T cells or populations of T cells according to the invention can exhibit, for example, an improved antigen response or class I antigen response compared to a reference T cell or population of T cells. Preferably, the improvement or comparable improvement compared to the reference is demonstrated over a period or time course, as described herein above. T cells or populations of T cells according to the invention can optionally exhibit improved or increased CD40L expression, affinity for antigen-presenting cells, and / or cytokine production, e.g., cytotoxic activity as determined by a cell killing assay of cells expressing the T cell-recognized antigen, tumor, or cancer antigen, induction of dendritic cell maturation, or induction of dendritic cell cytokine production, in response to a cancer or tumor antigen or peptide, or a cancer peptide, antigenic peptide, or peptide fragment of a cancer or tumor antigen, or presented by a tumor in a cancer cell or tissue, and recognized or bound by a T cell or a heterologous TCR or CAR.

[0097] Compositions and Therapies The invention provides T cells or populations of T cells produced according to the methods of the invention.

[0098] The invention further provides a composition comprising a T cell or population of T cells produced according to the methods of the invention and a physiologically acceptable excipient.

[0099] The T cells or populations of modified T cells according to the present invention can be mixed with other reagents, such as buffers, carriers, diluents, preservatives, and / or pharmaceutically acceptable excipients. Pharmaceutical compositions suitable for administration (e.g., by injection) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions, which may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Suitable vehicles can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990. In some preferred embodiments, the modified T cells or populations of T cells according to the present invention can be formulated into pharmaceutical compositions suitable for intravenous infusion into an individual.

[0100] The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0101] The present invention provides a T cell or a population of T cells or a composition thereof produced according to the method of the present invention for use in adoptive therapy.Therefore, the modified T cell or a population of T cells can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally or by intravenous infusion.Preferably, the modified immunoresponsive cell can be administered intravenously or by intravenous infusion. Thus, the engineered T cells or population of T cells can be administered as a single dose or as two or more doses (multiple doses), and can be administered in a dose of any one of about 500 million to about 1 billion cells, about 2 billion cells, about 3 billion cells, about 4 billion cells, about 5 billion cells, about 6 billion cells, about 7 billion cells, about 8 billion cells, about 9 billion cells, about 10 billion cells, about 11 billion cells, about 12 billion cells, about 13 billion cells, about 14 billion cells, about 15 billion cells, about 16 billion cells, about 17 billion cells, about 18 billion cells, about 19 billion cells, about 20 billion cells, or about 21 billion cells.

[0102] The present invention provides a T cell or population of T cells, or a composition thereof, produced according to the method of the present invention for use in treating tumors and / or cancers. The tumors and / or cancers can be selected from lung cancer, non-small cell lung cancer (NSCLC), metastatic or advanced NSCLC, squamous NSCLC, adenocarcinoma NSCLC, adenosquamous NSCLC, large cell NSCLC, ovarian cancer, gastric cancer, urothelial carcinoma, esophageal cancer, esophagogastric junction cancer (EGJ), melanoma, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, oropharynx cancer, hypopharyngeal cancer, throat cancer, laryngeal cancer, tonsil cancer, tongue cancer, soft palate cancer, pharyngeal cancer, synovial sarcoma, and myxo-round cell liposarcoma (MRCLS). According to the present invention, the cancer can be selected from any one of breast cancer, metastatic breast cancer, liver cancer, renal cell carcinoma, synovial sarcoma, urothelial carcinoma or tumor, pancreatic cancer, colorectal cancer, metastatic stomach cancer, metastatic gastric cancer, metastatic liver cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic colorectal cancer, metastatic lung cancer, colorectal cancer or adenocarcinoma, lung cancer or adenocarcinoma, pancreatic cancer or adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and hematological malignancies. Optionally, the cancer and / or tumor can express MAGE-A4 or AFP or an antigenic peptide or peptide antigen of MAGE-A4 and / or comprising the amino acid sequence GVYDGREHTV, SEQ ID NO: 1 (MAGE-A4) or an antigenic peptide or peptide antigen of alpha-fetoprotein (AFP) and / or comprising the sequence FMNKFIYEI (SEQ ID NO: 2) or residues 158 to 166 from alpha-fetoprotein (AFP) (SEQ ID NO: 3).

[0103] treatment Preferably, the treatment comprises a reduction in the number of T cells or T cells in a population of T cells compared to a treatment comprising a reference T cell or population of T cells, as described herein above. (a) progression-free survival, (b) progression-free interval; (c) duration of response; (d) overall survival; (e) objective response or objective response rate; (f) overall response or overall response rate; (g) partial response or partial response rate; (h) complete response or complete response rate; (i) Disease stability rate or median disease stability (j) median progression-free survival; (k) Median progression-free time, (l) median duration of response, or (m) median overall survival; (n) Median objective response or median objective response rate; (о) Median overall response or median overall response rate, (p) median partial response or median partial response rate; (q) median complete response or median complete response; (r) Median disease stability rate or median disease stability to extend or improve or effectively extend or effectively improve.

[0104] "Overall survival" means subjects who remain alive for a defined period of time.

[0105] "Objective response rate" (ObRR) is the proportion of subjects with a predefined amount of tumor size reduction, optionally determined by the sum of the longest diameters (SLD) of target lesions or tumors, over a minimum period of time. "Overall response rate" (ORR) is defined as the proportion of subjects with a partial or complete response to therapy; stable disease is not included. ORR is generally defined as the sum of complete responses (CR) and partial responses (PR) over a specified period of time. "Progression-free survival" (PFS) means the time from treatment (or randomization) to first disease progression or death. "Time to progression" (TTP) is equivalent to PFS, except that it does not count patients who die from causes other than the cancer or tumor being treated. "Duration of response" (DoR) is the length of time a cancer, tumor, or lesion continues to respond to treatment without growth or dissemination. According to the present invention, DoR, TTP and PFS can be assessed by Response Evaluation Criteria in Solid Tumors (RECIST) or by CA-125 levels as determinants of progression.

[0106] According to the present invention, a "complete response" (CR) is determined when all target lesions or tumors are assessed or measured as having disappeared. A "partial response" (PR) is determined when, for example, a measurement of at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions or tumors is observed, when reference is made to a control or pretreatment comparator. A "progressive disease" (PD) is determined when, for example, a measurement of at least a 20% increase in the sum of the longest diameters (SLD) of target lesions or tumors is observed since the start of treatment or the presence of one or more new lesions, when reference is made to a control or pretreatment comparator. A "stable disease" (SD) is determined when, using the smallest SLD since the start of treatment as a reference, there is not a sufficient reduction or decrease in the sum of the longest diameters (SLD) of target lesions or tumors to qualify as a PR, and there is not a sufficient increase to qualify as a PD.

[0107] According to the present invention, (a) a kit comprising an effective amount of a T cell or population of T cells produced according to the methods of the invention, and a package insert containing instructions for using the T cells to treat or delay the progression of cancer and / or tumors in a subject; is provided.

[0108] The invention is further illustrated by reference to the following figures and examples. [Brief explanation of the drawings]

[0109] [Figure 1] Figure 1 shows the time points for addition of AKTi. Schematic diagram showing the time points when MK-2206 was added to T cell expansion cultures, with a comparison made between day 0 + 2 addition and day 2 only cultures. Day 0 addition occurs after bead addition and T cell activation, but before transduction / expansion. Day 2 addition occurs after transduction but before expansion. Addition to the media used in G-Rex supplementation does not disrupt the process. [Figure 2]Figure 1 shows T cell total nucleated cell (TNC) counts in response to MK-2206 for healthy donor material (HDM). HDM were expanded in 10 M G-Rex for 10 days. Cultures were treated with various concentrations of MK-2206 added at three different time points: day 0 (+D2), day 2. Total nucleated cell (TNC) counts were obtained 10 days after collection on an automated cell counter, ViCELL. [Figure 3] Figure 1 shows multiple healthy donor expansions at day 10 harvest after MK-2206 compound addition. Six healthy donor materials were expanded for 10 days in 10 M G-Rex according to the process described. Cultures were treated with MK-2206 at decreasing concentrations from 10 μM to 0.05 μM. Graph A details compounds added on day 0 (+day 2 media addition), while graph B details compounds added on day 2 only. Harvested cells were counted using a ViCELL. Results were displayed as fold change in expansion relative to untreated controls. Where replicate cultures were expanded, the mean ± SEM of two replicates is shown for each data point. [Figure 4] Figure 1 shows healthy donor memory phenotype distribution in the transduced CD8+ compartment. Healthy donor material was expanded for 10 days in 10 M G-Rex according to the process described. Cultures were treated with AKTi MK-2206 at multiple concentrations (μM) indicated on the x-axis. Compounds were added on days 0+2 or day 2 only. Recovered cells were analyzed by flow cytometry gating on the CD3+ / Dextramar+ / CD8+ compartment for memory phenotype markers (CCR7+ / -, CD45RA+ / -). [Figure 5] Figure 1 shows antigen-stimulated IFNγ secretion by T cells expanded in the presence of MK-2206. Expanded T cells were thawed from freezing, rested for 2 hours, and then co-cultured with an antigen-positive cell line (A375, seeded the day before) for 48 hours. Supernatants from stimulated T cell cultures were analyzed for cytokine release using an IFNγ ELISA. Each data point represents the mean ± SEM of three replicates. MK-2206 was added on days 0 + 2, 2. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0110] [Example 1] 1.1. Purpose The objective of the following study was to investigate a novel process for generating T cells in culture that could result in improved T cell function without penalizing T cell expansion, such that the resulting T cell population would have improved functional effectiveness in adoptive therapy and cancer treatment. In particular, it was particularly desirable for the process to increase the proportion of a relatively undifferentiated T cell population within the functional CD8+ compartment, with minimal or no negative impact on T cell expansion, T cell viability, transduction, and CD8 frequency in the final population, and also with improved cytokine secretion in response to antigen-specific activation. It was anticipated that such a cell population would have improved antitumor activity (cytotoxic cell killing activity and cytokine production) as judged by effector function in response to antigen, and would demonstrate improved persistence as judged by prolonged survival in response to antigen stimulation. In addition, the T cells would have improved persistence and memory formation, survival, and antigen-stimulated survival. The process incorporates the use of AKT inhibitors, exemplified here by MK-2206, a small molecule inhibitor of protein kinase B (AKT) that acts upstream of the glycolytic pathway. Improved T cell expansion was achieved when MK-2206 was added on day 2 after T cell activation on day 0 and transduction on day 1, and analysis showed improved functionality of the resulting T cell population, as described below.

[0111] 1.2. T Cell Isolation, Expansion, and Culture All cells were expanded and transduced using a stationary expansion system (G-Rex® Expandable). Cryopreserved leukopheresis starting material from healthy donors or cancer patients was thawed, washed, activated with CD3 / CD28 magnetic Dynabeads, and the CD3+ positive fraction was magnetically separated. Starting leukopheresis material was similar between healthy donors. CD3+ cells ranged from 44 to 64%, and all had an increased purity of 79 to 87% after positive isolation using anti-CD3 / CD28 Dynabeads. CD3+ phenotyping identified a range of stem cell memory (SCM) markers (CCR7+ / CD45RA+) in the CD8+ compartment between individuals, ranging from 11.6 to 57%. Enriched CD3+ T cells were cultured at 1.5 x 10 in a G-Rex® Quiescent Cell Expansion System device in a final culture volume of 10% using TexMACS + 5% human AB serum (HABS) supplemented with 100 IU / mL IL-2. 6 TNC / cm 2 Cells were seeded at a density based on total nucleated cell (TNC) count. Transduction with a vector expressing a heterologous TCR (recognizing MAGE-A4) was performed 18-26 hours after the addition of CD3 / CD28 Dynabeads to the cells using a lentiviral (LV) vector at an MOI of 0.45. The final culture volume was then filled up to the final volume, which was performed 17-24 hours after transduction with TexMACS + 5% HABS + 500 IU / mL IL-2. The cells were then incubated at 37°C and 5% CO2 for an additional 8 days. MK-2206 was added to the medium at predetermined intervals; either on day 0, as a supplement to the medium used for seeding and day 2 supplementation, or only as a supplement to the medium used for day 2 supplementation. The compound concentrations varied for each experiment and were 10 μM, 7.5 μM, 5 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, and 0.05 μM, as shown in the following examples. T cells were harvested 10 days after seeding. After removing the beads, T cells were counted by automated cell analysis (using ViCELL). The harvested cells were then frozen for subsequent phenotyping and functional analysis as described. An overview of the process is shown in Figure 1.

[0112] 1.3 Antigen stimulation and cytokine release assay (IFNγ) The functionality of expanded T cells was assessed based on the level of cytokine release by expanded T cells after antigen stimulation with A375, a MAGE-A4-expressing cell line. A375 MAGE-A4-positive target cells were counted using an automated cell counter and seeded into 96-well U-bottom plates at 30,000 viable nucleated cells (VNCs) per well in a volume of 100 μL per well. After 1 day, T cell stimulation was performed and incubated overnight at 37°C and 5% CO2. Wells used as "T cell alone" controls received only 100 μL R10 medium (RPMI medium containing 10% FBS and 1% penicillin / streptomycin). After overnight target cell incubation, harvested and frozen T cells were thawed in a water bath at 37°C and washed with R10 medium. Cells were incubated at 37°C and 5% CO2 for 2 hours at 2 × 10 6 The cells were rested in R10 medium at a density of 150,000 VNCs / mL. After 2 hours of rest, T cells were counted using an automated cell counter, and cultures were normalized to a transduction efficiency of 35% for all donors by combining the calculated amounts of transduced and non-transduced VNCs for each condition. The normalized T cells were seeded at 150,000 VNCs / well in a 100 μL volume onto plates containing A375 target cells seeded the day before. The plates were returned to a 37°C, 5% CO2 incubator for 48 hours to allow T cell stimulation. After 48 hours of stimulation, the plates were centrifuged at 400G for 5 minutes, and the supernatant was transferred to a new 96-well plate. The supernatant was stored at -20°C.

[0113] ELISA was performed using human IFNγ by ELISA. Standard controls were prepared for the plate to a maximum concentration of 10,000 pg / mL for IFNγ, serially diluted 1:2 to generate a standard curve for each assay. Colorimetric readout was analyzed at OD450 (BMG LABTECH FLUOstar Omega plate reader).

[0114] [Example 2] 2.1.T cell expansion analysis Healthy donor material (leukapheresis material containing leukocytes isolated from blood donor samples) was processed for culture in a static expansion (G-Rex device) after the process described above for a duration of 10 days. Cells were cultured with MK-2206 (1.25, 2.5, 5.0, or 10 μM) added to the culture on days 0 + 2 or on day 2 only (with medium addition on day 2). At harvest, total nucleated cell (TNC) counts were performed using an automated cell counter (ViCELL) to provide expansion data, and flow cytometry was performed to determine phenotype. Harvested cultures were frozen and subsequently thawed for functional assessment, antigen-stimulated cytokine release assays (1.3 above).

[0115] The data (Figure 2) showed increased expansion in the presence of relatively low concentrations of MK-2206 when compared to untreated controls. All time points for MK-2206 addition showed reduced expansion at 10 μM. With the exception of 10 μM, addition on day 2 alone resulted in improved TNC yields. Day 0 (+2) consistently resulted in lower yields.

[0116] [Example 3] 3.1. Extended titration and expanded analysis MK-2206 concentrations were tested in further titrations from 0.05 to 10 μM to fully determine the optimal dose, and both day 0 (+2) and day 2 alone were evaluated to determine the optimal time of addition. Again, expansion, phenotypic, and cytokine production data were evaluated. Four additional donors were tested at various concentrations from 10 μM to 0.05 μM to provide a full titration range in this study. All donors were tested in 10 M G-Rex following the described process and collected on day 10. Expansion data examined the time points of MK-2206 addition; day 0 + 2 and day 2 alone.

[0117] The time of MK-2206 addition had an effect on expansion. Day 0+2 did not show a consistent response. Donor variability was observed across all concentrations tested, with limits of a 1.5-fold increase in expansion and a 0.15-fold decrease in expansion. Peak expansion was at the lowest concentrations (0.05 and 0.1 μM), where all donors responded similarly to or 0.5-fold better than untreated controls (Figure 3A). Day 2 addition had no negative effect on expansion. Donor variability was present; however, all conditions resulted in responses similar to or better than controls (≥1-2-fold). Day 2 addition resulted in peak expansion for three donors at 0.5 μM, before expansion began to decline in a dose-dependent response (0.05-0.25 μM). The expansion response appeared to plateau above 0.5 μM, with some donor variability (Figure 3B). Overall, day 2 additions resulted in a significantly better response to expansion than day 0+2 additions.

[0118] [Example 4] 3.4.T cell phenotypic analysis Flow cytometry was performed on healthy donor material recovered on day 10 from the described process to determine memory phenotype markers of cultured cells using CCR7 and CD45RA staining.

[0119] The data in Figure 4 show that cells cultured with MK-2206 exhibited an expanded CCR7+CD45RA+ population at harvest when added on days 0+2 and day 2 only, compared with untreated controls. The concentration of addition had no significant effect on increasing CCR7+CD45RA+. The increase in these stem cell-like markers was more reflective of the day 0 starting leukoreduced material (Figure 4D0), suggesting that addition likely maintains this population by slowing differentiation.

[0120] As shown (Figure 4), MK-2206 has an effect on memory phenotype markers (CCR7 / CD45RA), and an increase in CD62L expression is also observed, thereby increasing the population of these less terminally differentiated double-positive SCM-like cells that are less susceptible to functional exhaustion and are more likely to persist with longer in vivo survival, resulting in longer-lasting and more durable immune responses. This increase in the CCR7+ population is indicative of an increase in stem cell-like and central memory markers and a consequent decrease in the CCR7-CD45RA-, i.e., more terminally differentiated effector memory (EM) population, which has the potential for exhaustion and reduced T cell survival and proliferation in vivo. The EMRA population generally remains similar to untreated controls across all concentrations tested. The expansion in the CCR7+ population indicates that cell culture with MK-2206 is generating "fitter" T cells with the capacity for survival and persistence, which has functional benefits for the T cell population, particularly in the context of adoptive therapy and cancer treatment.

[0121] SCM T cells are CCR7+ / CD45RA+ "stem memory cells" (T SCM SCM T cells are relatively non-terminally differentiated T cells with a high capacity for long-term responses and self-renewal and survival. They share similarities with naive T cells, also expressing high amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibiting many functional attributes characteristic of memory cells. SCM T cells are relatively non-terminally differentiated T cells with a high capacity for long-term responses and self-renewal and survival. CM T cells are also known as "central memory T cells" (T CM CM T cells are memory subpopulations commonly found in lymph nodes and peripheral circulation. EM T cells are "effector memory T cells" (T EMThese memory T cells are CCR7- / CD45RA-, i.e., lack expression of CCR7 and CD45RA. They also have intermediate to high expression of CD44. These memory T cells lack lymph node homing receptors and are therefore found in the peripheral circulation and tissues; they are immediate response effector cells and suffer functional attrition due to their terminally differentiated state. EMRA cells (T EMRA ) are terminally differentiated effector memory cells (CCR7- / CD45RA+) that re-express CD45RA, a marker normally found on naive T cells.

[0122] [Example 5] 5.1. Cytokine production Expanded T cells derived from the T cell culture process described above were thawed and seeded in the presence of A375, a MAGE-A4 positive cell line. For cytokine release assays, target and effector cells were co-cultured for 48 hours, and then the supernatant was collected and analyzed for IFNγ levels by ELISA. A non-transduced control was also included in the assay as an additional control. All samples were normalized to a 35% transduction rate.

[0123] The level of IFNγ produced by antigen-stimulated T cells was examined as an indicator of T cell population functionality. MK-2206 was evaluated against untreated controls for each donor. The response in IFNγ production after antigen stimulation was, to some extent, donor-dependent. Increasing MK-2206 concentrations also appeared to decrease cellular cytokine production (>5 μM). The greatest increase was seen with the addition of MK-2206 on day 2 (Figure 5).

[0124] array GVYDGREHTV, (SEQ ID NO: 1), MAGE A4 peptide

[0125] FMNKFIYEI (SEQ ID NO: 2) alpha-fetoprotein (AFP) peptide or residues 158-166 from SEQ ID NO: 3

[0126] Human alpha-fetoprotein SEQ ID NO: 3

[0127] [ka]

[0128] SEQ ID NO: 4; MAGE A4 TCR α chain, CDRs are bold and underlined

[0129] [ka]

[0130] SEQ ID NO: 5: MAGE A4 TCR α chain coding sequence

[0131] [ka]

[0132] SEQ ID NO: 6; (MAGE A4 TCR β chain) CDRs are bold and underlined

[0133] [ka]

[0134] SEQ ID NO: 7 (MAGE A4 TCR β chain coding sequence)

[0135] [ka]

[0136] SEQ ID NO: 8; (MAGE A4 TCR α chain variable region) 136AA-CDR is bold and underlined MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYT VSPFSN LRWYKQDTGRGPVSLTI LTFSEN TKSNGRYTATLDADTKQSSLHITASQLSDSASYI CVVSGGTDSWGKLQF GAGTQVVVTPD

[0137] SEQ ID NO: 9; (MAGE A4 TCR β chain variable region) 133AA-CDR is bold and underlined MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQT KGHDR MYWYRQDPGLGLRLIYY SFDVKD INKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYF CATSGQGAYEEQFF GPGTRLTVLE

[0138] SEQ ID NO: 10; CDR1 MAGE A4 TCR alpha chain VSPFSN

[0139] SEQ ID NO: 11; CDR2 MAGE A4 TCR alpha chain LTFSEN

[0140] SEQ ID NO: 12; CDR3 MAGE A4 TCR alpha chain CVVSGGTDSWGKLQF

[0141] SEQ ID NO: 13; CDR1 MAGE A4 TCR β chain KGHDR

[0142] SEQ ID NO: 14; CDR2 MAGE A4 TCR β chain SFDVKD

[0143] SEQ ID NO: 15; CDR3 MAGE A4 TCR β chain, CATSGQGAYEEQFF

[0144] Parent AFP TCR TRAV12-2 * 02 / TRAJ41 * 01 / TRAC alpha chain amino acid extracellular sequence (SEQ ID NO: 16)

[0145] [ka]

[0146] Parental AFP TCR alpha chain DNA sequence (SEQ ID NO: 17)

[0147] [ka]

[0148] Parental AFP TCR TRBV9 * 01 / TRBD2 / TRBJ2-7 * 01 / TRBC2 beta chain amino acid extracellular sequence (SEQ ID NO: 18)

[0149] [ka]

[0150] Parental AFP TCR β chain DNA sequence (SEQ ID NO: 19)

[0151] [ka]

[0152] Mutant AFP TCR (AFP TRAV12-2 * 02 / TRAJ41 * 01 / TRAC alpha chain amino acid extracellular sequence (SEQ ID NO: 20)

[0153] [ka]

[0154] Mutant AFP TCR TRBV9 * 01 / TRBD2 / TRBJ2-7 * 01 / TRBC2 beta chain amino acid extracellular sequence (SEQ ID NO: 21)

[0155] [ka]

[0156] DRGSQS(αCDR1), AFP TCR, SEQ ID NO: 22

[0157] IYSNGD(αCDR2), AFP TCR, SEQ ID NO: 23

[0158] AVNSDSGYALNF(αCDR3), AFP TCR, SEQ ID NO: 24

[0159] SGDLS(βCDR1), AFP TCR, SEQ ID NO: 25

[0160] YYNGEE (βCDR2), AFP TCR, SEQ ID NO: 26

[0161] ASSLGGESEQY(βCDR3), AFP TCR, SEQ ID NO: 27

[0162] DRGSQA(αCDR1), AFP TCR, SEQ ID NO: 28

[0163] AVNSDSSYALNF(αCDR2), AFP TCR, SEQ ID NO: 29

[0164] AVNSDSGVALNF(αCDR2), AFP TCR, SEQ ID NO: 30

[0165] AVNSQSGYALNF(αCDR2), AFP TCR, SEQ ID NO: 31

[0166] AVNSQSGYSLNF(αCDR2), AFP TCR, SEQ ID NO: 32

[0167] AVNSQNGYALNF(αCDR2), AFP TCR, SEQ ID NO: 33

[0168] DRGSFS(αCDR1), AFP TCR, SEQ ID NO: 34

[0169] DRGSYS(αCDR1), AFP TCR, SEQ ID NO: 35

[0170] AVNSQSSYALNF(αCDR2), AFP TCR, SEQ ID NO: 36

[0171] [ka]

[0172] [ka]

[0173] VLLSNPTSG, CD8α CDR1, SEQ ID NO: 39

[0174] YLSQNKPK, CD8α CDR2, SEQ ID NO: 40

[0175] LSNSIM, CD8α CDR3, SEQ ID NO: 41 The inventions described in the original claims of this application are listed below. [Invention 1] (a) activating an isolated population of T cells; (b) culturing the T cells; (c) modifying the T cells to express at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR), preferably by transducing the T cells with a nucleic acid or vector encoding at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR); (d) adding an inhibitor of AKT (AKT inhibitor) to the modified T cells; (e) culturing the modified T cell population to expand and / or grow the cells or cell population; (f) optionally, harvesting and / or cryopreserving the modified T cell population. A method for producing modified T cells, comprising: [Invention 2] 2. The method of claim 1, wherein the T cells are enriched for the CD3+ fraction. [Invention 3] 3. The method of claim 1 or 2, wherein said activation stimulates said population of T cells to proliferate. [Invention 4] 4. The method according to claim 3, wherein said activation is by the addition of an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof, optionally attached to removable beads. [Invention 5] 5. The method according to any one of claims 1 to 4, wherein the modification is carried out prior to or simultaneously with activation. [Invention 6] 5. The method according to any one of claims 1 to 4, wherein the modification is carried out after activation, preferably 18 to 26 hours after activation. [Invention 7] 7. The method according to any one of claims 1 to 6, wherein the AKT inhibitor is added after the modification, preferably 17 to 24 hours after the modification. [Invention 8] 8. The method according to any one of claims 1 to 7, wherein the AKT inhibitor is selected from the group consisting of allosteric inhibitors, competitive ATP inhibitors, inhibitors of the interaction between AKT and phospholipids, and inhibitors of the phosphorylation of molecules downstream of AKT, preferably PRAS40, ribosomal S6, and TSC2. [Invention 9] 9. The method of claim 8, wherein the allosteric inhibitor is selected from ARQ092, ARQ751, BAY1125976, or MK-2206. [Invention 10] 9. The method of claim 8, wherein the competitive ATP inhibitor is selected from afuresertib (GSK2110183), GSK2141795, GSK690693, ipatasertib (GDC-0068), LY2780301, triciribine (TCN-PM; VD-0002), AZD5363, or CCT128930. [Invention 11] The method according to invention 8, wherein the inhibitor of the interaction between AKT and phospholipids is perifosine (D-21266, KRX0401). [Invention 12] 12. The method according to any one of Inventions 1 to 11, wherein the AKT inhibitor is added at a concentration of 0.10 μM to 10 μM, preferably 0.5 μM. [Invention 13] the modified T cell or population of T cells produced in the presence of the AKT inhibitor, (i) a population of T cells or modified T cells produced in the absence of an AKT inhibitor, or (ii) a population of T cells or modified T cells produced in the presence of an AKT inhibitor, wherein the AKT inhibitor is added prior to modification and / or 24 hours or less after activation. Compared to (a) T cells expressing both CD45RA+ and CCR7+; (b) T cells that are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+; (c)T SCM T cells, which are stem memory T cells; (d) T cells having a memory phenotype, preferably a CD8 memory phenotype 13. The method of any one of claims 1 to 12, wherein the method has an increased or high relative proportion of any one or more of: [Invention 14] the altered population of T cells produced in the presence of the AKT inhibitor, (i) a population of T cells or modified T cells produced in the absence of an AKT inhibitor, or (ii) a population of T cells or modified T cells produced in the presence of an AKT inhibitor, wherein the AKT inhibitor is added prior to transduction and / or 24 hours or less after stimulation. Compared to (a) persistence in vivo, preferably measured over 8-14 days after in vivo injection; (b) expansion from seeding to harvesting; (c) durability of response or durable response rate (DRR); (d) antigen-induced interferon-γ production; (e) T cell survival rate or lifespan or viability percentage; (f) T cell effector function, preferably cytotoxicity 14. The method according to any one of claims 1 to 13, wherein the method has improved or increased levels of any one or more of: [Invention 15] 15. A population of modified T cells produced according to the method of any one of claims 1 to 14. [Invention 16] 16. A composition comprising a population of modified T cells according to invention 15 and a physiologically acceptable excipient. [Invention 17] 17. A population of modified T cells according to invention 15 or a composition according to invention 16 for use in adoptive therapy. [Invention 18] 17. A population of modified T cells according to invention 15 or a composition according to invention 16 for use in the treatment of cancer and / or tumors. [Invention 19] 15. A kit comprising a population of modified T cells produced according to the method of any one of inventions 1 to 14, and a package insert containing instructions for using said T cells to treat or delay the progression of cancer and / or tumors in a subject.

Claims

1. (a) activating an isolated population of T cells; (b) culturing the T cells; (c) modifying the activated T cells to express at least one heterologous T cell receptor (TCR) or chimeric antigen receptor (CAR); (d) adding an inhibitor of AKT (AKT inhibitor) to the modified T cells 17 to 24 hours after the modification; (e) culturing the modified T cell population to expand and / or proliferate the cells or cell population. A method for producing modified T cells, comprising:

2. The method described in claim 1, wherein the T cells are modified by transducing the T cells with a nucleic acid or vector encoding the at least one TCR or CAR.

3. The method described in claim 1 or 2, further comprising the step of (f) recovering and / or cryopreserving the modified T cell population.

4. The method of any one of claims 1 to 3, wherein the T cells are enriched for the CD3+ fraction.

5. The method of any one of claims 1 to 4, wherein said activation stimulates said population of T cells to proliferate.

6. The method of claim 5, wherein the activation is by the addition of an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof.

7. The modification is performed 18 to 26 hours after activation.

7. The method according to any one of claims 1 to 6.

8. The method of any one of claims 1 to 7, wherein the AKT inhibitor is selected from the group consisting of an allosteric inhibitor, a competitive ATP inhibitor, an inhibitor of the interaction between AKT and phospholipids, and an inhibitor of the phosphorylation of a molecule downstream of AKT.

9. (a) the allosteric inhibitor is ARQ092, ARQ751, BAY1125976, or MK-2206; (b) the competitive ATP inhibitor is afuresertib (GSK2110183), GSK2141795, GSK690693, ipatasertib (GDC-0068), LY2780301, triciribine (TCN-PM; VD-0002), AZD5363, or CCT128930, or (c) the inhibitor of the interaction between AKT and phospholipids is perifosine (D-21266, KRX0401); The method of claim 8.

10. The method of any one of claims 1 to 9, wherein the AKT inhibitor is added at a concentration of 0.10 μM to 10 μM.

11. The method described in claim 10, wherein the AKT inhibitor is added at a concentration of 0.5 μM.

12. the modified T cell or population of T cells produced in the presence of the AKT inhibitor, (i) a population of T cells or modified T cells produced in the absence of an AKT inhibitor, or (ii) a population of T cells or modified T cells produced in the presence of an AKT inhibitor, wherein the AKT inhibitor is added prior to modification and / or no more than 24 hours after activation. Compared to (a) T cells that express both CD45RA+ and CCR7+; (b) T cells that are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+; (c) T SCM T cells that are stem memory T cells, (d) T cells with a memory phenotype 12. The method of claim 1, wherein the soluble fraction of ...

13. the altered population of T cells produced in the presence of the AKT inhibitor, (i) a population of T cells or modified T cells produced in the absence of an AKT inhibitor, or (ii) A population of T cells or modified T cells produced in the presence of an AKT inhibitor, wherein the AKT inhibitor is added prior to transduction and / or 24 hours or less after stimulation. Compared to (a) persistence in vivo; (b) expansion from seeding to harvesting; (c) durability of response or durable response rate (DRR); (d) antigen-induced interferon-γ production; (e) T cell survival or lifespan or viability percentage; (f) T cell effector function 13. The method of any one of claims 1 to 12, having improved or increased levels of any one or more of:

14. 14. A population of modified T cells produced according to the method of any one of claims 1 to 13.

15. 15. A composition comprising the population of modified T cells of claim 14 and a physiologically acceptable excipient.

16. 16. The population of modified T cells of claim 14 or the composition of claim 15 for use in adoptive therapy or in the treatment of cancer and / or tumors.

17. 14. A kit comprising a population of modified T cells produced according to the method of any one of claims 1 to 13 and a package insert containing instructions for using the T cells to treat or delay the progression of cancer and / or tumors in a subject.

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