Methods of treating cancer with engineered t cells

Abstract

This application relates to methods for treating cancer with engineered T cells. Novel adoptive immunotherapy compositions comprising co-cultured lentiviral vector-transferred autoantigen-presenting cells and T cells are provided herein, along with methods for using them in patient-specific combination immunotherapy for the treatment of cancer and other diseases and conditions.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680044596.2 entitled “Method for treating cancer with engineered T cells”. The original application was PCT international application PCT / US2016 / 037120 filed on June 12, 2016, which entered the Chinese national phase on January 29, 2018.

[0002] Cross-reference of related applications

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 175,003, filed June 12, 2015, pursuant to Section 119(e) of 35 USC, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This application relates to the field of cancer, and more particularly to compositions comprising autologous antigen-presenting cells co-cultured with autologous T cells transduced with chimeric antigen receptor (CAR), and methods for use in patient-specific combination immunotherapy, wherein the autologous antigen-presenting cells are transduced via a lentiviral vector expressing a cancer transcript with a patient-specific mutation. Background Technology

[0005] Cancer is one of the deadliest threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after cardiovascular disease, accounting for about a quarter of all deaths. Solid tumors are responsible for the majority of these deaths. Despite significant advances in drug treatments for some cancers, the overall 5-year survival rate for all cancers has only improved by about 10% in the past 20 years. Cancer, or malignant tumors, metastasizes and grows rapidly and uncontrollably, making treatment extremely difficult. One of the challenges of modern cancer treatment is the amount of time that passes between cancer biopsy and diagnosis and the patient's effective treatment. During this time, the patient's tumor may grow unimpeded, allowing the disease to progress further before treatment can be implemented. This negatively impacts the prognosis and outcome of cancer.

[0006] Chimeric antigen receptors (CARs) are hybrid molecules comprising three basic units: (1) an extracellular antigen-binding motif, (2) a linker / transmembrane motif, and (3) an intracellular T-cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2 (4): e23621). The antigen-binding motif of a CAR typically follows a single-chain variable fragment (scFv) (the minimal binding domain of an immunoglobulin (Ig) molecule). Alternative antigen-binding motifs have also been engineered, such as receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or γ-δ T cells) are also under development (Brown CE et al., Clin Cancer Res. 2012;18(8):2199–209; Lehner M et al., PLoS One. 2012; 7(2): e31210). Important work remains to be done on defining the most active T cell populations for transduction with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0007] The linker motif in a CAR can be a relatively stable domain, such as the constant domain of IgG, or designed as an extended flexible linker. Structural motifs (e.g., those derived from the constant domain of IgG) can be used to extend the scFv binding domain away from the T cell plasma membrane surface. This could be important for certain tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialiacoganglioside GD2; unpublished observations by Orentas et al.). To date, the signaling motif used in CARs has always included the CD3-ζ chain, as this core motif is a key signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and a CD28 transmembrane sequence. This motif has also been used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; 183(9):5563–74). With the advent of new technologies, the activation of T cells with beads linked to anti-CD3 and anti-CD28 antibodies and the presence of the classic “signal 2” from CD28 no longer need to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays, and they did not provide a significant advantage over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7):1165–74; Kochenderfer JN et al., Blood. 2012; 119 (12):2709–20). The clinical success of second-generation CD28 / CD3-ζ CD19-specific CARs (Lee DW et al., American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and the CD137 / CD3-ζ signaling modality (Porter DL et al., N Engl J Med. 2011; 365(8): 725–33) confirms this.In addition to CD137, other members of the tumor necrosis factor receptor superfamily (such as OX40) can also provide important and persistent signals in CAR-transduced T cells (Yvon E et al., Clin Cancer Res. 2009;15(18):5852–60). The culture conditions for culturing CAR T cell populations are also important.

[0008] Current challenges in the broader and more effective improvement of CAR therapy for cancer involve the lack of compelling targets. Creating binders for cell surface antigens is currently relatively easy, but finding cell surface antigens that are tumor-specific while not sparing normal tissue remains a significant challenge. One possible approach to enabling CAR-expressing T cells to have higher target cell specificity is to use a combinatorial CAR approach. In one system, CD3-ζ and CD28 signaling units are separated between two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has lower affinity, thus requiring conjugation first of the alternative CAR for the full activity of the second (Lanitis E et al., Cancer Immunol Res. 2013;1(1):43–53; Kloss CC et al., Nat Biotechnol. 2013;31(1):71–5). A second challenge for generating single scFv-based CARs as immunotherapeutic agents is tumor cell heterogeneity. At least one research group has developed a CAR strategy for glioblastoma in which effector cell populations simultaneously target multiple antigens (HER2, IL-13Ra, EphA2) in order to avoid the growth of target antigen-negative populations (Hegde M et al., Mol Ther. 2013;21(11):2087–101).

[0009] T-cell-based immunotherapy has emerged as a new frontier in synthetic biology; multiple promoters and gene products have been devised to guide these highly potent cells into the tumor microenvironment, where T cells can both evade negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with AP1903 has demonstrated a way in which a powerful switch that can control a T-cell population can be pharmacologically activated (Di Stasi A et al., N Engl J Med. 2011;365(18):1673–83). The generation of effector T-cell populations that generate negatively regulated immunity against transforming growth factor β through decoy receptor expression further demonstrates that effector T cells can be engineered to achieve optimal antitumor activity (Foster AE et al., J Immunother. 2008;31(5):500–5).

[0010] Therefore, although CAR appears to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacles to the clinical application of this CAR-based technology to date have been limited to the in vivo expansion of CAR+ T cells, the rapid disappearance of cells after infusion, disappointing clinical activity, and the excessively long time between using such CAR+ T cells for cancer diagnosis and timely treatment.

[0011] Therefore, there is an urgent and long-standing need in the art to discover compositions and methods for treating cancer using CAR-based therapies that can exhibit patient-specific expected therapeutic properties without the aforementioned drawbacks.

[0012] The present invention addresses these needs by providing compositions comprising autoantigen-presenting cells / T cells transduced with co-cultured lentiviral vectors and methods for using them in patient-specific combination therapies for the treatment of cancer and other diseases and / or conditions.

[0013] In particular, the present invention, as disclosed and described herein, provides a composition comprising autologous antigen-presenting cells transduced with a lentiviral vector expressing a patient-specific tumor-encoded mutant cancer antigen, said cells being co-cultured with autologous T cells transduced with a lentiviral vector expressing a chimeric antigen receptor (CAR), having or not having one or more lentivirally expressed tumor biopsy and peripheral blood-derived tumor antigen T cell receptors transduced into a therapeutic T cell population to generate an active patient-specific anti-tumor T cell population that can be directly infused back into the patient to promote in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer. Summary of the Invention

[0014] This article provides a novel adoptive immunotherapy composition comprising autoantigen-presenting cells and T cells transduced with a co-cultured lentiviral vector, and a method for using it in patient-specific combination immunotherapy for the treatment of cancer and other diseases and conditions.

[0015] Therefore, in one aspect, this document provides lentiviral vectors expressing patient-specific mutated cancer antigens, lentiviral vectors expressing natural T-cell receptors (TCRs), lentiviral vectors expressing tumor-specific reactive T-cell TCR transcripts, and lentiviral vectors expressing chimeric antigen receptors (CARs), as well as host cells (e.g., T cells) expressing mutated cancer antigens, natural T-cell receptors, T-cell TCR transcripts, and receptors, and nucleic acid molecules encoding mutated cancer antigens, natural T-cell receptors, T-cell TCR transcripts, and receptors. Methods for using the disclosed lentiviral vectors expressing patient-specific mutated cancer antigens, lentiviral vectors expressing natural T-cell receptors (TCRs), lentiviral vectors expressing tumor-specific reactive T-cell TCR transcripts, and lentiviral vectors expressing chimeric antigen receptors (CARs), host cells, and nucleic acid molecules are also provided, for example, to treat cancer in a subject.

[0016] In one aspect, an adoptive immunotherapy composition is provided comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autoantigen-presenting cells expressed with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0017] In one implementation, the autoantigen-presenting cells are derived from autologous dendritic cells, B cells, a mixture thereof, or lymphocytes derived from peripheral blood.

[0018] In one embodiment, an adoptive immunotherapy composition is provided in which autologous patient-specific T cells containing a natural T cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens, to generate an active population of patient-specific autologous anti-tumor T cells capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0019] In one embodiment, an adoptive immunotherapy composition is provided, wherein a patient-derived tumor antigen is identified by patient biopsy and nucleotide sequencing to identify mutant RNA transcripts within a mutanome.

[0020] In one embodiment, an adoptive immunotherapy composition is provided, wherein the autologous anti-tumor T cell population comprises autologous antigen-presenting cells (APCs) containing patient-specific dendritic cells or B cells or a mixture of them or peripheral blood-derived lymphocytes.

[0021] In another embodiment, an adoptive immunotherapy composition is provided, wherein the autologous anti-tumor T cell population comprises autologous antigen-presenting cells (APCs) containing viable patient-specific autologous B cells immortalized with Epstein-Barr virus (EBV), wherein the immortalization step includes culturing the autologous B cells together with a cell culture supernatant containing EBV. In one embodiment, commercial services for producing such viable patient-specific autologous B cells immortalized with EBV include, for example, but not limited to, Applied Biologic Material, ABM, Inc., (https: / / www.abmgood.com / EBV-Cell-Immortalization.html). In one embodiment, the EBV-immortized B cell line comprises cell lines conventionally used in the art, including but not limited to the EBV-immortized B cell line B95-8 (ATCC CRL-1612, or EBV-containing supernatant (ATCC-BR14-92)).

[0022] On the other hand, adoptive immunotherapy compositions are provided comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors, wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0023] In one embodiment, an adoptive immunotherapy composition is provided, wherein a tumor-specific T-cell receptor (TCR) is first identified by co-culturing antigen-presenting cells (APCs) transduced with one or more lentiviral vectors expressing patient-derived tumor antigens with HLA-compatible or patient-specific T cells.

[0024] In one implementation, the autoantigen-presenting cells are derived from autologous dendritic cells, B cells, a mixture thereof, or lymphocytes derived from peripheral blood.

[0025] In one embodiment, an adoptive immunotherapy composition is provided, wherein the tumor-specific T-cell receptor (TCR) is HLA-compatible or patient-specific.

[0026] In one embodiment, an adoptive immunotherapy composition is provided, wherein autologous patient-specific T cells containing a patient-specific tumor-specific T-cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens, to generate an active population of patient-specific autologous anti-tumor T cells capable of recognizing the tumor-specific T-cell receptor (TCR) and capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0027] In one embodiment, an adoptive immunotherapy composition is provided, wherein patient-derived tumor antigens are identified by patient biopsy and nucleotide sequencing to identify mutant RNA transcripts within a mutant group. In one embodiment, next-generation sequencing is used for nucleotide sequencing.

[0028] In one embodiment, an adoptive immunotherapy composition is provided, wherein the autologous anti-tumor T cell population comprises autologous antigen-presenting cells (APCs) containing patient-specific dendritic cells or B cells or a mixture of these or peripheral blood-derived lymphocytes.

[0029] In some embodiments, an adoptive immunotherapy composition is provided in which an active patient-specific autologous anti-tumor T cell population is generated within one, three, five, seven, ten, fourteen, twenty-one, or one month after tumor biopsy, and wherein the active patient-specific autologous anti-tumor T cell population can be infused back into a patient with cancer and is capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer.

[0030] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the CAR comprises at least one extracellular antigen-binding domain, at least one adapter domain, at least one transmembrane domain and at least one intracellular signal transduction domain.

[0031] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one single-chain variable fragment of an antibody that binds to the antigen.

[0032] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one heavy chain variable region of an antibody that binds to the antigen.

[0033] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein at least one extracellular antigen-binding domain of the CAR, at least one intracellular signal transduction domain of the CAR, or both are connected to a transmembrane domain via a linker or spacer-binding domain.

[0034] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the extracellular antigen-binding domain of the CAR is preceded by a lead peptide.

[0035] In certain embodiments of the two aspects mentioned above, adoptive immunotherapy compositions are provided, wherein the extracellular antigen-binding domain of the CAR targets an antigen comprising CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0036] In certain embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the extracellular antigen-binding domain of the CAR comprises an anti-CD19 scFV antigen-binding domain, an anti-CD20 scFV antigen-binding domain, an anti-CD22 scFV antigen-binding domain, an anti-ROR1 scFV antigen-binding domain, an anti-TSLPR scFV antigen-binding domain, an anti-mesothelin scFV antigen-binding domain, an anti-CD33 scFV antigen-binding domain, an anti-CD38 scFV antigen-binding domain, an anti-CD123 (IL3RA) scFV antigen-binding domain, an anti-CD138 scFV antigen-binding domain, an anti-BCMA (CD269) scFV antigen-binding domain, an anti-GPC2 scFV antigen-binding domain, an anti-GPC3 scFV antigen-binding domain, an anti-FGFR4 scFV antigen-binding domain, an anti-c-Met scFV antigen-binding domain, and an anti-PMSA domain. The scFV antigen-binding domain, the anti-glycolipid F77 scFV antigen-binding domain, the anti-EGFRvIII scFV antigen-binding domain, the anti-GD-2 scFV antigen-binding domain, the anti-NY-ESo-1TCR scFV antigen-binding domain, the anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0037] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the CAR's linker or spacer domain is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

[0038] In certain embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the CAR further comprises a transmembrane domain comprising a transmembrane domain selected from the following proteins: α, β, or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, or any combination thereof.

[0039] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the at least one intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0040] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

[0041] In some embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0042] In certain embodiments of the two aspects mentioned above, an adoptive immunotherapy composition is provided, wherein the at least one co-stimulatory domain comprises functional signal transduction domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137) or any combination thereof.

[0043] On the one hand, this article provides isolated nucleic acid molecules encoding cancer antigens with patient-specific mutations, isolated nucleic acid molecules encoding natural T cell receptors (TCRs), isolated nucleic acid molecules encoding tumor-specific reactive T cell TCR transcripts, or isolated nucleic acid molecules encoding chimeric antigen receptors (CARs).

[0044] In one aspect of using CARs for active patient-specific autologous anti-tumor T cell populations, CARs are modified to express or contain detectable biomarkers for diagnosis, monitoring and / or prediction of treatment outcomes (e.g., progression-free survival in cancer patients) or for monitoring the progression of such treatment.

[0045] In one embodiment of CAR for use with an active patient-specific autologous anti-tumor T cell population, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector (e.g., a viral vector). The vector may be a DNA vector, RNA vector, plasmid vector, granular vector, herpesvirus vector, measles virus vector, lentiviral vector, adenovirus vector or retroviral vector, baboon endogenous virus (BaEV), or a combination thereof.

[0046] In some embodiments of CAR for use with active patient-specific autologous anti-tumor T cell populations, the lentiviral vector is pseudotyped with different viral glycoproteins (GPs), including, but not limited to, non-retroviral GPs such as, murine leukemia virus [MLV-A], GP164, gibberish leukemia virus [GALV], RD114, feline endogenous retrovirus-derived GPs and vesicular stomatitis virus [VSV], measles virus, avian plague virus [FPV], Ebola virus [EboV], and lymphocytic choriomeningitis virus [LCMV], as well as their chimeric variants, including, but not limited to, chimeric GPs encoding the extracellular domain and transmembrane domain of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP.

[0047] In some embodiments of CAR for use with active patient-specific autologous anti-tumor T cell populations, the vector also contains a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0048] In another embodiment of CAR for use in an active patient-specific autologous anti-tumor T cell population, the CAR-expressing vector can be further modified to include one or more operational elements to control CAR T cell expression, or to eliminate CAR-T cells via a suicide switch. The suicide switch may include, for example, a drug that induces apoptosis in a signaling cascade or induces cell death. In a preferred embodiment, the CAR-expressing vector can be further modified to express enzymes such as thymidine kinase (TK) or cytosine deaminase (CD).

[0049] In another aspect of using CAR for an active patient-specific autologous anti-tumor T cell population, a host cell comprising a nucleic acid molecule encoding the CAR is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from the subject. In one embodiment, the host cell is a CD8+ T cell.

[0050] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of active patient-specific autologous antitumor T cells from a person suffering from cancer, wherein the cancer is a refractory cancer unresponsive to one or more chemotherapy agents. Cancers include hematopoietic system cancers, myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other blood cancers and solid tumors, or any combination thereof.

[0051] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of active patient-specific autologous antitumor T cells from a person suffering from cancer, wherein the cancer includes blood cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma or Hodgkin lymphoma) or multiple myeloma, or any combination thereof.

[0052] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of active patient-specific autologous antitumor T cells from a person suffering from cancer, wherein said cancer includes adult epithelial cancers, including oropharyngeal cancers (tongue, mouth, pharynx, head, and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile ducts, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0053] On the other hand, a method for preparing active patient-specific autologous anti-tumor CAR-containing T cells is provided. The method comprises transducing T cells with a vector or nucleic acid molecule encoding: i) one or more patient-specific mutant cancer antigens; ii) one or more patient-specific and tumor-specific TCRs; and iii) one or more chimeric antigen receptors (CARs) or any combination thereof that specifically bind to antigens, thereby preparing active patient-specific autologous anti-tumor CAR-containing T cells.

[0054] On the other hand, a method is provided for generating a population of RNA-engineered T cells, comprising introducing in vitro transcribed RNA or synthetic RNA encoding nucleic acid molecules of the following: i) one or more patient-specific mutated cancer antigens; ii) one or more patient-specific and tumor-specific TCRs; and iii) one or more chimeric antigen receptors (CARs) or any combination thereof, thereby generating an active population of patient-specific autologous anti-tumor T cells capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0055] On the other hand, a pharmaceutical composition is provided comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autoantigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0056] In another aspect, a pharmaceutical composition is provided comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of recognizing the tumor-specific T cell receptors (TCRs) and capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0057] In one embodiment, a pharmaceutical composition is provided, wherein the T cells are T cells from a person with blood cancer.

[0058] In another embodiment, a pharmaceutical composition is provided in which the blood cancer is leukemia or lymphoma.

[0059] In another embodiment, a pharmaceutical composition is provided, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML).

[0060] In another embodiment, a pharmaceutical composition is provided, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

[0061] In another embodiment, a pharmaceutical composition is provided, wherein the hematologic cancer is multiple myeloma. In yet another embodiment, a pharmaceutical composition is provided, wherein human cancers include adult epithelial cancers, including oropharyngeal cancers (tongue, mouth, pharynx, head, and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile ducts, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and cancers of the brain and other nervous systems, or any combination thereof.

[0062] On the other hand, a method is provided for treating mammals suffering from diseases, disorders, or conditions associated with elevated tumor antigen expression, the method comprising administering to a subject a pharmaceutical composition comprising an antitumor-effective amount of an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous antitumor T cell population capable of promoting in vivo expansion and persistence of patient-specific antitumor T cells in a patient-specific manner, leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer.

[0063] On the other hand, a method is provided for treating mammals suffering from diseases, disorders, or conditions associated with elevated tumor antigen expression, the method comprising administering a pharmaceutical composition to a subject comprising an antitumor-effective amount of an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous antitumor T cell population capable of recognizing the tumor-specific T cell receptors (TCRs), the active patient-specific autologous antitumor T cell population being capable of being directly infused back into the patient to promote in vivo expansion and persistence of patient-specific antitumor T cells in a patient-specific manner, leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer.

[0064] In some implementations, a method is provided herein in which T cells have been pre-selected by expressing specific activation or memory-related surface markers.

[0065] In some embodiments, a method is provided herein in which T cells and dendritic cells are derived from a hematopoietic stem cell donor, and wherein said method is performed in the case of hematopoietic stem cell transplantation.

[0066] In another aspect, a method is provided for generating a persistent population of genetically engineered, active patient-specific autologous anti-tumor T cells in a person diagnosed with cancer. In one embodiment, the method includes administering one or more active patient-specific autologous anti-tumor T cell populations as described herein to a patient in need, wherein the persistent population of the active patient-specific autologous anti-tumor T cell population or a population of progeny of the T cells persists in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years after administration.

[0067] In one embodiment, the progeny T cells in the human body include memory T cells. In another embodiment, the T cells are autologous T cells.

[0068] In all aspects and embodiments of the methods described herein, one or more compositions comprising the active patient-specific autologous anti-tumor T cell populations disclosed herein may be used to treat, prevent, or improve the aforementioned cancers, diseases, disorders, or conditions associated with elevated tumor antigen expression.

[0069] On the other hand, a kit is provided for preparing compositions comprising active patient-specific autologous anti-tumor T cell populations as described above or for the prevention, treatment or improvement of any cancer, disease, disorder or condition associated with elevated tumor antigen expression in the subjects described above, the kit comprising a container containing any one or any combination of the above-described nucleic acid molecules, vectors, host cells or compositions, and instructions for using the kit.

[0070] It should be understood that active patient-specific autologous anti-tumor T cell populations, lentiviral vectors expressing patient-specific mutant cancer antigens, lentiviral vectors expressing natural T cell receptors (TCRs), lentiviral vectors expressing tumor-specific reactive T cell TCR transcripts, and lentiviral vectors expressing chimeric antigen receptors (CARs), as well as host cells (e.g., T cells) expressing mutant cancer antigens, natural T cell receptors, T cell TCR transcripts, and receptors, and nucleic acid molecules encoding mutant cancer antigens, natural T cell receptors, T cell TCR transcripts, and receptors, are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of this disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0071] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0072] Figure 1 A first exemplary method for treating cancer is described, wherein T cells designated for immunotherapy (reinfused back into the patient) are transduced with CAR-expressing LVs and stimulated by their native TCRs to recognize patient-specific mutant proteins identified by next-generation sequencing.

[0073] Figure 2 A second exemplary method for treating cancer is described, wherein T cells designated for immunotherapy (reinfused back into the patient) are transduced with LVs expressing CARs and TCR sequences derived from tumor biopsy or blood, and stimulated by transcripts expressing DCs identified by next-generation sequencing of the tumor. Detailed Implementation Plan

[0074] definition

[0075] Unless the context clearly indicates otherwise, nouns without quantifiers as used herein refer to both the singular and plural forms. For example, the term "antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." The term "comprising" as used herein means "including." Therefore, "comprising antigen" means "including antigen" without excluding other elements. The phrase "and / or" means "and" or "or." It should be further understood that, unless otherwise stated, any and all base sizes or amino acid sizes and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and provided for descriptive purposes. While many methods and materials similar to or equivalent to those described herein may be used, specific suitable methods and materials are described below. In case of conflict, this specification (including the interpretation of terms) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not restrictive. For ease of reference to various embodiments, the following interpretations of terms are provided:

[0076] When referring to measurable values ​​(e.g., quantity, short duration, etc.), the term “about” means to cover a variation of + / -20%, + / -10%, or more preferably + / -5%, or + / -1%, or even more preferably + / -0.1% from a particular value, because such variation is suitable for performing the disclosed method.

[0077] Unless otherwise stated, the technical terms used herein shall be used in accordance with their usual usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, Oxford University Press, 1999; Kendrew et al., The Encyclopedia of Molecular Biology, 1994, Blackwell Science Ltd.; Robert A. Meyers, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995; and other similar references.

[0078] This invention relates to compositions and methods for treating cancers, including but not limited to hematologic malignancies and solid tumors. This invention relates to patient-specific, tumor-specific strategies for adoptive cell transfer via transduced T cells expressing chimeric antigen receptors (CARs).

[0079] More specifically, this invention relates to lentiviral vectors expressing patient-specific mutant cancer antigens, lentiviral vectors expressing natural T-cell receptors (TCRs), lentiviral vectors expressing tumor-specific reactive T-cell TCR transcripts, and lentiviral vectors expressing the chimeric antigen receptor (CAR) provided herein, as well as host cells (e.g., T cells) expressing mutant cancer antigens, natural T-cell receptors, T-cell TCR transcripts, and receptors, and nucleic acid molecules encoding mutant cancer antigens, natural T-cell receptors, T-cell TCR transcripts, and receptors. Methods for using the disclosed lentiviral vectors expressing patient-specific mutant cancer antigens, lentiviral vectors expressing natural T-cell receptors (TCRs), lentiviral vectors expressing tumor-specific reactive T-cell TCR transcripts, and lentiviral vectors expressing chimeric antigen receptor (CAR) molecules, host cells, and nucleic acid molecules are also provided, for example, to treat cancer in a subject.

[0080] Surprisingly and unexpectedly, the inventors have now discovered that an active anti-tumor population of T cells is more effective when expressed in conjunction with the expression of a chimeric antigen receptor (CAR) (by selecting a population of natural T cells or molecular clones and transferring the TCR via a lentiviral vector). CARs surprisingly and unexpectedly allow for the persistence of a population of T cells carrying the tumor-specific TCR, achieved by stimulating this population upon encountering a self-antigen (e.g., CD19) that the patient can tolerate losing and which also serves to provide a stimulating signal to a therapeutic cell population not present in the tumor tissue itself. Such an active patient-specific anti-tumor T cell population, as described herein, can be directly infused back into the patient to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells, thereby leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer in a patient-specific manner.

[0081] Therefore, in its broadest sense, the novelty of this adoptive immunotherapy lies in using a lentiviral vector to identify the patient's TCR by transducing APCs with a tumor-specific mutated gene and then culturing them with the patient's T cells. This involves sequencing and identifying the mutated antigen in the patient, then expressing the mutated protein in the APCs via LV, and co-culturing T cells and identifying the patient's TCR. Mutant group-specific TCRs and T cells can then be isolated and characterized. Additionally, a CAR is then added to enhance the immune response (IR). The distinguishing feature is that the CAR is not the primary immunotherapeutic agent, but rather used to enhance a highly specific TCR response. It enhances the IR in two distinct ways: first, by providing additional signals to T cells to expand and survive in vivo; and second, by targeting immunosuppressive cell antigens.

[0082] On the other hand, the novelty of this adoptive immunotherapy lies in using a lentiviral vector to identify patient-derived tumor-specific TCRs by transducing APCs with tumor-encoded mutant genes using LVs and then culturing them with patient cells. This involves sequencing and identifying mutant antigens from the patient, then expressing the mutant protein in the APCs via LVs, and co-culturing patient T cells to identify mutant group-specific TCRs. On another front, CARs are used to enhance the immune response to tumors mediated by a therapeutic T cell population. This enhancement of the immune response occurs in at least three ways. First, by providing additional signals for T cells to expand and survive in vivo, CARs allow for the persistence of a therapeutic T cell population carrying tumor-specific TCRs, achieved by stimulating this T cell population upon encountering an autoantigen (e.g., CD19) that the patient can tolerate losing and which also serves to provide a stimulating signal to a therapeutic cell population not present in the tumor tissue itself. Second, CARs can target cell types other than tumors that mediate immunosuppression. For example, if CD19-expressing B cells are present in tumor lesions and also mediate anti-tumor effects, a second benefit of using a CAR-expressing tumor-specific T cell population is the removal of immunosuppressive cell populations. Thirdly, CARs target immunosuppressive populations distal to the tumor, i.e., immunosuppressive populations existing in another compartment of the body. For example, using CARs that target bone marrow-derived suppressor cells (MDSCs) that may be present in the tumor lesion itself or in regional lymph nodes or bone marrow.

[0083] The following is a detailed description of the CARs that can be used with the active patient-specific autologous anti-tumor T cell populations disclosed herein, including descriptions of their extracellular domains, transmembrane domains and intracellular domains, as well as additional descriptions of the CARs, antibodies using the disclosed CARs and their antigen-binding fragments, conjugates, nucleotides, expression, vectors and host cells, therapeutic methods, compositions and kits.

[0084] A. Chimeric antigen receptor (CAR)

[0085] The CAR disclosed in this paper comprises at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0086] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. CARs are characterized by their ability to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner, and by utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition endows CAR-expressing T cells with the ability to recognize antigens independently of antigen processing, thereby bypassing major tumor escape mechanisms. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0087] As disclosed herein, the intracellular T-cell signaling domain of a CAR may include, for example, a T-cell receptor signaling domain, a T-cell co-stimulatory signaling domain, or both. The T-cell receptor signaling domain refers to a portion of the CAR containing the intracellular domain of the T-cell receptor, such as, but not limited to, the intracellular portion of the CD3ζ protein. The co-stimulatory signaling domain refers to a portion of the CAR containing the intracellular domain of a co-stimulatory molecule, which is a cell surface molecule required for an effective lymphocyte response to an antigen, other than an antigen receptor or its ligand.

[0088] 1. Extracellular domains

[0089] In one implementation, the CAR used in the active patient-specific autologous anti-tumor T cell population disclosed herein includes a target-specific binding element, which is also referred to as an antigen-binding domain or portion. The choice of domain depends on the type and number of ligands defining the surface of the target cell. For example, an antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a specific disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in a CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0090] In one implementation, a CAR can be engineered to target a specific tumor antigen by engineering a desired antigen-binding domain that specifically binds to antigens on tumor cells. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T-cell-mediated one. The choice of antigen-binding domain will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muco-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. The tumor antigens disclosed herein are included only by way of example. This list is not exclusive, and other examples will be apparent to those skilled in the art.

[0091] In one implementation, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignancy. Malignant tumors express a number of proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, CD22, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0092] Tumor antigens can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells; instead, they are expressed on normal cells even when immune tolerance to the antigen cannot be induced. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. When the immune system is immature and unable to respond, TAAs can be antigens expressed on normal cells during embryonic development, or they can be antigens that are normally present at very low levels in normal cells but expressed at much higher levels on tumor cells.

[0093] Non-limiting examples of TSA or TAA include the following: differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (PmeI 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other major protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / Cycophilic C-related protein, TAAL6, TAG72, TLP, and TPS.

[0094] In a preferred embodiment, the antigen-binding domain of the CAR partially targets antigens including, but not limited to, the following: CD19, CD20, CD22, ROR1, mesothelin, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc.

[0095] Depending on the desired antigen to be targeted, a CAR can be engineered to include an appropriate antigen-binding domain that is specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding domain and incorporated into the CAR.

[0096] In one exemplary embodiment, the antigen-binding domain of the CAR partially targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 scFV, wherein the nucleic acid sequence of the anti-CD19 scFV comprises the sequence shown in SEQ ID NO:27. In one embodiment, the anti-CD19 scFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:28. In another embodiment, the anti-CD19 scFV portion of the CAR comprises the amino acid sequence shown in SEQ ID NO:28.

[0097] In one aspect of the invention, a CAR capable of binding to a non-TSA or non-TAA is provided, which includes, for example but not limited to, antigens derived from the Retroviridae family (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), picoronavir viruses (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae family, herpesvirus family [e.g., herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesvirus], poxvirus family (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0098] In another aspect of the invention, a CAR is provided that is capable of binding to antigens from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. Specifically, CARs are provided that can bind to antigens from infectious bacteria (such as Helicobacter pylori, Legionella pneumophila), mycobacterial strains (such as Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, or Mycobacterium gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof).

[0099] 2. Transmembrane domain

[0100] In the CARs disclosed herein for use in active patient-specific autologous anti-tumor T cell populations, the CARs contain one or more transmembrane domains fused to the extracellular domains of the CAR.

[0101] In one implementation, an isolated nucleic acid molecule is provided, wherein the encoded adapter domain is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

[0102] In one implementation, an isolated nucleic acid molecule is provided, wherein the encoded adapter domain is derived from and linked to the extracellular domain of the transmembrane domain.

[0103] In some cases, the binding of such a domain to the transmembrane domain of the same or different surface membrane proteins can be avoided by selecting a transmembrane domain or by amino acid substitution, in order to minimize interactions with other members of the receptor complex.

[0104] The transmembrane domain can be derived from natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. Specifically, the transmembrane region used in this invention can be derived from (i.e., containing at least the following transmembrane regions): the α, β, or ζ chain of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. Optionally, short oligopeptide or polypeptide linkers, preferably 2 to 10 amino acid lengths, can form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine duplexes provide a particularly suitable linker.

[0105] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO:11. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:12. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:12.

[0106] In some cases, the transmembrane domain of the CAR includes a CD8.α. hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO:13. In one embodiment, the CD8 hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14. In another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO:14.

[0107] Not intended to limit any particular mechanism of action, the possible reasons for the enhanced therapeutic function associated with the exemplary CARs used in active patient-specific autologous anti-tumor T cell populations as disclosed herein are, for example, but not limited to: a) improved lateral movement within the plasma membrane allowing for more efficient signal transduction; b) superior location within plasma membrane microstructural domains (e.g., lipid rafts) and the ability to interact better with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane away from inhibitory or downregulatory interactions (e.g., less proximity to or interaction with phosphatases such as CD45) by preferential movement; and d) superior assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0108] In one embodiment of the active patient-specific autologous anti-tumor T cell population disclosed herein, non-limiting exemplary transmembrane domains (including TNFRSF16 and TNFRSF19 transmembrane domains) for use with the CAR disclosed herein can be used to obtain the TNFRSF transmembrane domains and / or linker or spacer subdomains disclosed in the applicant’s co-pending provisional patent application No. 62 / 239,509 entitled CHIMERIC ANTIGEN RECEPTORS AND METHODSOF USE, filed October 9, 2015, and assigned to Miltenyi Biotech Technology, Inc., with material number LEN_015PRO, including, in particular, those other TNFRSF members listed in the tumor necrosis factor receptor superfamily as listed in Table 1.

[0109] 3. Spacer subdomain

[0110] In the CARs disclosed herein for use with active patient-specific autologous anti-tumor T cell populations, a spacer domain may be arranged between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide used to link the TNFRSF transmembrane domain to the extracellular domain and / or the TNFRSF transmembrane domain to the intracellular domain. The spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0111] In several embodiments, the connector may include spacer elements that, in their presence, increase the size of the connector, thereby increasing the distance between the effector molecule or detectable biomarker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and are included in U.S. Patent Nos. 7,964,5667,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, and 5,530,0 The following are listed in U.S. Patent Publications 20110212088 and 20110070248, and are incorporated herein by reference in their entirety: 97, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248.

[0112] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signal transduction into the cell. Examples of amino acids expected to promote binding include cysteine ​​(a charged amino acid) at possible glycosylation sites, as well as serine and threonine, and these amino acids can be used as amino acids constituting the spacer domain.

[0113] As a spacer domain, all or a portion of amino acids 118 to 178 (SEQ ID NO: 15) of the hinge region of CD8.α (NCBI RefSeq: NP.sub.--001759.3), amino acids 135 to 195 of CD8.β (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq.NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq.NP.sub.--006130.1) can be used. Furthermore, as a spacer domain, a portion of the constant region of the antibody H chain or L chain (CH1 region or CL region, e.g., a peptide having the amino acid sequence shown in SEQ ID NO.: 16) can be used. Additionally, the spacer domain can be a synthetically produced sequence.

[0114] Furthermore, in a CAR, a signal peptide sequence can be attached to the N-terminus. Signal peptide sequences are present at the N-terminus of many secretory and membrane proteins and are 15 to 30 amino acids in length. Since many protein molecules that serve as intracellular domains possess signal peptide sequences, the signal peptide can be used as a signal peptide for a CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:6.

[0115] 4. Intracellular domains

[0116] The cytoplasmic domains or other intracellular signaling domains of a CAR are responsible for activating at least one of the normal effector functions of the immune cells in which the CAR has been implanted. The term "effector function" refers to a specific function of the cell. For example, the effector functions of a T cell can be lytic activity or helper activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specific function. While the entire intracellular signaling domain can often be used, in many cases it is not necessary to use the entire strand. Regarding the use of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the complete strand as long as it transduces effector function signals. Therefore, the term "intracellular signaling domain" means any truncated portion of an intracellular signaling domain that is sufficient to transduce effector function signals.

[0117] Preferred examples of intracellular signal transduction domains for use in CARs include cytoplasmic sequences of T-cell receptors (TCRs) and common receptors that work together to initiate signal transduction upon antigen-receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0118] It is known that the signal generated by the TCR alone is insufficient to fully activate T cells and a second signal or co-stimulatory signal is also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that induce antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0119] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that function in a stimulatory manner may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAM (immunoreceptor tyrosine-based activation motif).

[0120] Examples of primary cytoplasmic signaling sequences containing ITAM specifically used in the CARS disclosed herein include those derived from TCR ζ (CD3 ζ), FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAM include polypeptides having the following sequences: amino acids 51 to 164 of CD3 ζ (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of Fc.ε.RI.γ (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of Fc.ε.RI.β (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3 δ (NCBI RefSeq: NP.sub.--000723.1), and amino acids 128 to 171 of CD3.ε (NCBI RefSeq: NP.sub.--000723.1). Amino acids 153 to 207 of RefSeq: NP.sub.--000724.1, amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub._001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub._000617.1), amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub._001806.2), and their variants that have the same function as these peptides. The amino acid numbers based on the NCBIRefSeq ID or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling sequence derived from CD3ζ.

[0121] In a preferred embodiment, the intracellular domain of the CAR can be designed to contain a CD3-ζ signaling domain itself, or combined with any other desired cytoplasmic domains available in the case of a CAR. For example, the intracellular domain of the CAR may contain a portion of the CD3ζ chain and a co-stimulatory signaling region. A co-stimulatory signaling region refers to a portion of the CAR containing the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule, in addition to an antigen receptor or its ligand, required for an effective lymphocyte response to an antigen. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific, non-limiting examples of such co-stimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), and amino acids 4-1BB of CD137. Amino acids 214 to 255 of NCBIRefSeq: NP.sub.--001552.2, amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), as well as their variants that have the same function as these peptides. Therefore, although the disclosure herein primarily uses 4-1BB as an example of a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of this disclosure.

[0122] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked together in a random or specified order. Optionally, short oligopeptide or polypeptide linkers (preferably between 2 and 10 amino acid lengths) can form links. Glycine-serine duplexes provide particularly suitable linkers.

[0123] In one embodiment, the intracellular domain is designed to include a CD3-ζ signal transduction domain and a CD28 signal transduction domain. In another embodiment, the intracellular domain is designed to include a CD3-ζ signal transduction domain and a 4-1BB signal transduction domain. In yet another embodiment, the intracellular domain is designed to include a CD3-ζ signal transduction domain and signal transduction domains of both CD28 and 4-1BB.

[0124] In one embodiment, the intracellular domains in the CAR are designed to include a 4-1BB signal transduction domain and a CD3-ζ signal transduction domain, wherein the 4-1BB signal transduction domain contains the nucleic acid sequence shown in SEQ ID NO:17 and the CD3-ζ signal transduction domain contains the nucleic acid sequence shown in SEQ ID NO:19.

[0125] In one embodiment, the intracellular domains in the CAR are designed to include a 4-1BB signal transduction domain and a CD3-ζ signal transduction domain, wherein the 4-1BB signal transduction domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:18 and the CD3-ζ signal transduction domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20.

[0126] In one embodiment, the intracellular domains in the CAR are designed to include a 4-1BB signal transduction domain and a CD3-ζ signal transduction domain, wherein the 4-1BB signal transduction domain contains the amino acid sequence shown in SEQ ID NO:18 and the CD3-ζ signal transduction domain contains the amino acid sequence shown in SEQ ID NO:20.

[0127] 5. Additional description of CAR

[0128] The scope of this invention also explicitly includes functional portions of CARs used in the active patient-specific autologous anti-tumor T cell populations disclosed herein. When referring to the use of CAR, the term "functional portion" means any part or fragment of one or more CARs disclosed herein that retains the biological activity of the CAR (parental CAR) to which it is a part. Functional portions include, for example, those portions of CARs that retain, to a similar degree, or to the same degree, or to a greater degree, the ability to recognize target cells or to detect, treat, or prevent disease, as in relation to the parental CAR. Referring to the parental CAR, the functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parental CAR.

[0129] The functional moiety may contain additional amino acids at the amino or carboxyl terminus, or at both ends, which are not found in the amino acid sequence of the parental CAR. Desiredly, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. More desirously, the additional amino acids enhance the biological activity compared to the biological activity of the parental CAR.

[0130] Included within the scope of this disclosure are functional variants of the CARs disclosed herein. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, and that retains the biological activity of its variant CAR. Functional variants include, for example, those variants of the CARs (parental CARs) described herein that retain the ability to recognize target cells to a degree of similarity, identicalness, or greater than that of the parent CAR. Referring to a parent CAR, a functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98%, or more identity with the parent CAR in terms of amino acid sequence.

[0131] The functional variant may, for example, comprise the amino acid sequence of the parental CAR having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parental CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parental CAR.

[0132] The amino acid substitutions in CARs are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include such substitutions in which an amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of one acidic / negatively charged polar amino acid for another (e.g., Asp or Glu), the substitution of one amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), the substitution of one basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), the substitution of one uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), the substitution of one amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr, and Val), the substitution of one amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0133] CARs can consist essentially of a specific amino acid sequence as described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0134] CARs (including functional parts and functional variants) can have any length, that is, they can contain any number of amino acids, provided that the CAR (or its functional part or functional variant) retains its biological activity, such as the ability to specifically bind to an antigen, detect diseased cells in mammals, or treat or prevent diseases in mammals. For example, a CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids.

[0135] CARs (including the functional portions and functional variants of this invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, etc. Dihydroindole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0136] CARs (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated, by means of, disulfide bridges.

[0137] CARs (including their functional moieties and functional variants) can be obtained by methods known in the art. CARs can be prepared by any suitable method for preparing peptides or proteins. Suitable methods for de novo synthesis of peptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and US Patent 5,449,752. Methods for generating chimeric antigen receptors, including T cells containing such receptors, and their uses (e.g., for the treatment of cancer) are known in the art and further described herein (see, for example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp. 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; Tumaini et al., Cytotherapy, 15, 1406-1417, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety. For example, nucleic acid molecules encoding the disclosed chimeric antigen-binding receptor may be included in an expression vector (e.g., a lentiviral vector) for transducing host cells (e.g., T cells) to prepare the disclosed CAR.In some implementations, the method of using chimeric antigen receptors includes isolating T cells from a subject, transducing the T cells with an expression vector encoding the chimeric antigen receptor (e.g., a lentiviral vector), and administering CAR-expressing T cells to the subject for treatment, for example, for treating tumors in the subject.

[0138] B. Antibody-antigen binding fragments

[0139] One embodiment also provides a CAR for the active patient-specific autologous anti-tumor T cell population disclosed herein, CAR-expressing T cells, antibodies that specifically bind to one or more antigens disclosed herein, or their antigen-binding domains or portions thereof. As used herein, "CAR-expressing T cells" or "CAR T cells" refers to T cells that express CAR and have antigen specificity determined by, for example, a targeting domain derived from an antibody of the CAR.

[0140] As used herein, “antigen-binding domain” can include antibodies and their antigen-binding fragments. The term “antibody” is used herein in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and their antigen-binding fragments, provided they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art that retain binding affinity for antigens, as well as their variants and fragments.

[0141] "Monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, meaning that individual antibodies comprising that population are identical except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. In some instances, monoclonal antibodies are produced by a single clone of a cell or its progeny that has been transfected with B lymphocytes or nucleic acids that have encoded the variable regions of the light and heavy chains of an antibody (or its antigen-binding fragment). In some instances, monoclonal antibodies are isolated from an object. Monoclonal antibodies may have conserved amino acid substitutions that have little effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, see Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0142] Typically, immunoglobulins have heavy chains (H) and light chains (L) linked together by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). There are five main heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0143] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, for example, Kindt et al., Kuby Immunology, 6.sup.th ed., WH Freeman and Co., p. 91 (2007)). In several embodiments, the variable regions of the heavy and light chains are combined to specifically bind the antigen. In other embodiments, only the variable region of the heavy chain is required. For example, naturally occurring camel antibodies consisting only of the heavy chain are functional and stable in the absence of the light chain (see, for example, Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The reference to “VH” or “VH” refers to the variable region of the antibody heavy chain, including the variable region of antigen-binding fragments such as Fv, scFv, dsFv, or Fab. The term "VL" refers to the variable domain of the antibody light chain, including the variable domains of Fv, scFv, dsFv, or Fab.

[0144] The variable regions of the light and heavy chains contain "framework" regions interrupted by three hypervariable regions (also known as "complementarity-determining regions" or "CDRs") (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the frame regions of different light or heavy chains are relatively conserved within species. The frame regions of antibodies (i.e., the combined frame regions of constitutive light and heavy chains) are used to locate and arrange CDRs in three-dimensional space.

[0145] CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of many well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs on each strand are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are usually identified by the strand to which a particular CDR is located. Therefore, VH CDR3 is the CDR3 derived from the variable domain of the heavy chain of the antibody, while VL CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0146] An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize homologous antigens, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modifying a complete antibody or antigen-binding fragments synthesized de novo using recombinant DNA methods (see, for example, Kontermann and Dubel (eds.), Antibody Engineering, Vol. 1–2, 2nd ed., Springer Press, 2010).

[0147] Single-chain antibodies (scFvs) are genetically engineered molecules containing one or more VH and VL domains of antibodies linked together by suitable peptide linkers to form a single-chain molecule for gene fusion (see, for example, Bird et al., Science, 242:423 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:58795883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in scFvs is generally not decisive for scFvs. Therefore, scFv with two possible arrangements (VH-structural domain-joint structural domain-VL-structural domain; VL-structural domain-joint structural domain-VH-structural domain) can be used.

[0148] In dsFv, the heavy and light chain variable chains have been mutated to introduce disulfide bonds, thereby stabilizing chain association. Also included are bispecific antibodies, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker too short to pair between the two domains on the same chain, thus forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444 6448, 1993; Poljak et al., Structure, 2:1121 1123, 1994).

[0149] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0150] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be recombinantly generated, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy and light chains, as described in Huse et al., Science 246:1275-1281 (1989) (which is incorporated herein by reference). These and other methods for preparing, for example, chimeric, humanized, CDR-transplanted, single-chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, see above, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0151] "Antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Antibody competitive assays are known, and exemplary competitive assays are provided herein.

[0152] A “humanized” antibody or antigen-binding fragment comprises a human framework region and one or more core-receiving regions (CDRs) derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDR is referred to as the “donor,” while the human antibody or antigen-binding fragment providing the framework is referred to as the “recipient.” In one embodiment, all CDRs are derived from donor immunoglobulins in a humanized immunoglobulin. Constant regions are not required to be present, but if present, they can be substantially identical to constant regions of human immunoglobulins, for example, with at least about 85% to 90%, such as about 95% or higher identity. Thus, apart from the possible CDRs, all portions of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding portions of the natural human antibody sequence.

[0153] A "chimeric antibody" is an antibody that comprises sequences derived from two different antibodies (which are typically from different species). In some instances, a chimeric antibody includes one or more CDRs and / or frame regions from one human antibody and CDRs and / or frame regions from another human antibody.

[0154] A “fully human antibody” or “human antibody” is an antibody that includes a sequence from (or derived from) the human genome, but excludes sequences from another species. In some embodiments, a human antibody includes the CDR, framework region, and Fc region (if present) from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for generating antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, for example, Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0155] Antibodies can have one or more binding sites. If there are more than one binding site, the binding sites can be the same or different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0156] Methods for testing the ability of an antibody to bind to any functional part of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assay (see, for example, Janeway et al., see below, U.S. Patent Application Publication No. 2002 / 0197266A1 and U.S. Patent No. 7,338,929).

[0157] In addition, CARs, CAR-expressing T cells, antibodies, or their antigen-binding portions may contain detectable markers such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0158] C. Conjugates

[0159] Any number of methods known to those skilled in the art can be used to conjugate CARs for the active patient-specific autologous anti-tumor T cell populations disclosed herein, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof specific to one or more antigens disclosed herein to reagents such as effector molecules or detectable biomarkers. Both covalent and non-covalent linkages can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable biomarker is covalently linked to an antibody or antigen-binding fragment specifically binding to one or more antigens disclosed herein. Those skilled in the art will understand that a variety of effector molecules and detectable biomarkers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive reagents, etc. 125 I, 32 P, 14 C 3 H and 35 S and other markers, target portions, and ligands, etc.

[0160] The choice of a specific effector molecule or detectable biomarker depends on the specific target molecule or cell and the desired biological effect. Thus, for example, an effector molecule could be a cytotoxin used to induce the death of a specific target cell, such as a tumor cell.

[0161] Methods for attaching effector molecules or detectable biomarkers to antibody or antigen-binding fragments vary depending on the chemical structure of the effector. Peptides typically contain multiple functional groups; for example, carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can react with suitable functional groups on the antibody to result in binding of the effector molecule or detectable biomarker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching any of several known adapter molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). The adapter can be any molecule used to link the antibody or antigen-binding fragment to the effector molecule or detectable biomarker. The adapter is capable of forming a covalent bond with both the antibody or antigen-binding fragment and the effector molecule or detectable biomarker. Suitable adapters are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon adapters, heterocyclic carbon adapters, or peptide adapters. When the antibody or antigen-binding fragment and effector molecule or detectable biomarker are peptides, the linker can be connected to the constituent amino acid (e.g., to cysteine ​​via a disulfide bond) or to the α-carbon amino and carboxyl groups of the terminal amino acid via its side groups.

[0162] In several embodiments, the connector may include spacer elements that, in their presence, increase the size of the connector, thereby increasing the distance between the effector molecule or detectable biomarker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and include those listed in: U.S. Patent Nos. 7,964,5667,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5 U.S. Patent Publications 530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and U.S. Patent Publications 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker in the intracellular environment releases an effector molecule or detectable biomarker from the antibody or antigen-binding fragment. In other embodiments, the linker is not cleavable and releases the effector molecule or detectable biomarker, for example, through antibody degradation. In some embodiments, the linker may be cleaved by a cleaving agent present in the intracellular environment (e.g., within lysosomes, endosomes, or caveoles). The linker may be, for example, a peptide linker, which is cleaved by intracellular peptidases or proteases (including, but not limited to, lysosomal proteases or endosomal proteases). In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, or 1 to 15 amino acids long. Proteases may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug from the target cell (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, peptide linkers that can be cleaved by the thiol-dependent protease cathepsin B (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers) may be used. Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference. In one specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0164] In other embodiments, the cleavable adapter is pH-sensitive, meaning it is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive adapters are hydrolyzable under acidic conditions. For example, acid-labile adapters that are hydrolyzable in lysosomes (e.g., hydrazones, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used (see, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.). Such adapters are relatively stable under neutral pH conditions (e.g., in blood) but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond (see, for example, U.S. Patent No. 5,622,929).

[0165] In other embodiments, the connector is cuttable under reducing conditions (e.g., a disulfide connector). A variety of disulfide connectors are known in the art, including, for example, those that can be formed using: SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (edited by CW Vogel, Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008). See also U.S. Patent No. 4,880,935.

[0166] In other specific embodiments, the connector is a malonic acid ester connector (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl connector (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0167] In other embodiments, the connector is non-cuttable and releases effector molecules or detectable biomarkers through antibody degradation (see U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0168] In several embodiments, the adapter is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the adapter in the sample of the conjugate is cleaved. Whether the adapter is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the target adapter with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary adapters that can be used in conjugates are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0169] In several embodiments, conjugates are provided of CARs, CAR-expressing T cells, antibodies or their antigen-binding moieties with one or more small molecule toxins (e.g., calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene, and CC1065) and toxin-active derivatives of these toxins.

[0170] Maytansine compounds suitable for use as the toxin fraction of maytansine alkaloids are well known in the art and can be isolated from natural sources by known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthesized using known methods to prepare maytanol and maytanol analogues. Maytansine alkaloids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (US Patent No. 3,896,111). Subsequently, it was found that certain microorganisms also produce maytansine alkaloids, such as maytanol and C-3 maytanol ester (US Patent No. 4,151,042). Synthetic maytanol and its derivatives and analogues are disclosed, for example, in the following U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each incorporated herein by reference. Conjugates containing maytansine alkaloids, methods of their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Publications 5,208,020, 5,416,064, 6,441,163 and European Patent EP 0425 235 B1, the contents of which are expressly incorporated herein by reference.

[0171] Other toxins may be used in conjunction with CARs, CAR-expressing T cells, antibodies, or their antigen-binding portions. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saponins, and calciferine, as well as botulinum toxins A through F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Toxins of consideration also include variants of the toxin (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401).

[0172] Saponins are toxins derived from the saponaria officinalis plant that disrupt protein synthesis by partially inactivating the 60S region of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, these toxins lack a specific mechanism for cell entry and therefore require binding to antibody or antigen-binding fragments that recognize cell surface proteins, which are then internalized for efficient cellular uptake.

[0173] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxins used as immunotoxins are mutated to reduce or eliminate nonspecific toxicity. The CRM107 mutant, possessing complete enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0174] Ricin is an agglutinin RCA60 derived from castor beans (Ricinus communis) (castor seeds). For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Based on their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972), ricin (RCA) is referred to as RCA. 60 and RCA 120 It exists in two forms. The A chain is responsible for inactivating protein synthesis and killing cells. The B chain enables ricin to bind to galactose residues on the cell surface and facilitates the transport of the A chain into the cytoplasm (Olsnes et al., Nature 249:627-631, 1974 and US Patent No. 3,060,165).

[0175] Ribonucleases also bind to target molecules to act as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary nucleotoxins such as α-sarcin and restrictocin are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Galicin was first isolated from Micromonospora chinospora and is a member of the enediyne family of antitumor antibiotics that cause DNA double-strand breaks, leading to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic component of immunotoxins in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0176] Abrus precatorius toxins contain toxic lectins derived from the absinthes tree (Abrus precatorius). Mechanism of action: Abrus precatorius toxins a, b, c, and d have a molecular weight of approximately 63 to 67 kD and consist of two disulfide-linked polypeptide chains, A and B. Chain A inhibits protein synthesis; chain B (abrus precatorius toxin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0177] CARs for active patient-specific autologous anti-tumor T cell populations, CAR-expressing T cells, and monoclonal antibodies or antigen-binding fragments thereof specific to one or more antigens disclosed herein may also be conjugated to detectable biomarkers; for example, detectable biomarkers detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic endoscopy, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable biomarkers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent labels are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or their antigens bind to a detectable enzyme, detection can be achieved by adding additional reagents to produce a distinguishable reaction product. For example, in the presence of horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also bind to biotin and be detected by indirectly measuring the binding of avidin or avidin-streptin. It should be noted that avidin itself can be conjugated to enzymes or fluorescent labels.

[0178] CARs, CAR-expressing T cells, antibodies, or their antigen-binding fragments can be conjugated with paramagnetic reagents such as gadolinium. Paramagnetic reagents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pairs, binding sites of second antibodies, metal-binding domains, epitope tags).

[0179] CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated to radiolabeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect one or more antigens disclosed herein and cells expressing those antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabeling can be used therapeutically as a toxin for treating tumors, such as neuroblastoma. Examples of peptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0180] The methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, photographic film or a scintillation counter can be used to detect radioactive markers, and photodetectors can be used to detect fluorescent markers to detect emitted radiation. Enzyme markers are typically detected by providing a substrate to an enzyme and detecting the reaction products produced by the enzyme acting on the substrate, while colorimetric markers are detected simply by displaying a colored marker.

[0181] D. Nucleotides, expression, vectors, and host cells

[0182] Embodiments of the present invention also provide nucleic acids comprising nucleotide sequences encoding any CAR, antibody, or antigen-binding moiety thereof (including its functional portion and functional variants) described herein. The nucleic acids of the present invention may comprise nucleotide sequences encoding any leader sequence, antigen-binding domain, transmembrane domain, and / or intracellular T-cell signaling domain described herein.

[0183] In one embodiment, a separate nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, comprising, from the N-terminus to the C-terminus, at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signal transduction domain.

[0184] In one embodiment of a CAR for an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one single-stranded variable fragment of an antibody that binds to the antigen.

[0185] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to the antigen.

[0186] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain of the CAR contains at least one lipid-carrier-based antigen-binding antigen (anticalin) that binds to the antigen.

[0187] In one embodiment of CAR for use with an active patient-specific autologous anti-tumor T cell population, an isolated nucleic acid molecule is provided, wherein an encoded extracellular antigen-binding domain is linked to a transmembrane domain via a linker domain.

[0188] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0189] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain targets an antigen, said antigen including but not limited to: CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3TCR, or any combination thereof.

[0190] In certain embodiments of a CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain comprises an anti-CD19 scFV antigen-binding domain, an anti-CD20 scFV antigen-binding domain, an anti-CD22 scFV antigen-binding domain, an anti-ROR1 scFV antigen-binding domain, an anti-TSLPR scFV antigen-binding domain, an anti-mesothelin scFV antigen-binding domain, an anti-CD33 / IL3Ra scFV antigen-binding domain, an anti-CD38 scFV antigen-binding domain, an anti-CD123 (IL3RA) scFV antigen-binding domain, an anti-CD138 scFV antigen-binding domain, an anti-BCMA (CD269) scFV antigen-binding domain, an anti-GPC2 scFV antigen-binding domain, an anti-GPC3 scFV antigen-binding domain, an anti-FGFR4 scFV antigen-binding domain, and an anti-c-Met scFV antigen-binding domain. The scFV antigen-binding domain, the anti-PMSA scFV antigen-binding domain, the anti-glycolipid F77 scFV antigen-binding domain, the anti-EGFRvIII scFV antigen-binding domain, the anti-GD-2 scFV antigen-binding domain, the anti-NY-ESo-1 TCR scFV antigen-binding domain, the anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the above, or any combination thereof.

[0191] In one aspect of CARs for use with active patient-specific autologous anti-tumor T cell populations, the CARs provided herein also include a connector domain.

[0192] In one embodiment of a CAR for an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein an extracellular antigen-binding domain, an intracellular signal transduction domain, or both are linked to a transmembrane domain via a adapter domain.

[0193] In one embodiment of a CAR for an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded adapter domain is derived from the extracellular domain of CD8 and is linked to a transmembrane domain.

[0194] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the nucleic acid sequence encoding the transmembrane domain comprises a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0195] In one embodiment of a CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded transmembrane domain comprises an amino acid sequence containing at least one but no more than 10 modified amino acids, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0196] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain selected from the following proteins: the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or combinations thereof.

[0197] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0198] In one embodiment of the CAR disclosed herein, a separate nucleic acid molecule encoding the CAR is provided, wherein the encoded intracellular signal transduction domain is positioned on the C-terminal side relative to the CD3ζ intracellular domain.

[0199] In another embodiment of the CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, wherein at least one intracellular signaling domain encoded comprises a co-stimulatory domain, a major signaling domain, or a combination thereof.

[0200] In some other embodiments of CAR for use with active patient-specific autologous anti-tumor T cell populations, an isolated nucleic acid molecule encoding the CAR is provided, wherein at least one co-stimulatory domain encoding the CAR comprises a functional signal transduction domain comprising OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137) or a combination thereof.

[0201] In one embodiment of a CAR for use with an active patient-specific autologous anti-tumor T cell population, a separate nucleic acid molecule encoding the CAR is provided, which also includes a leader sequence or signal peptide sequence.

[0202] In some implementations, the nucleotide sequence can be codon-modified. Without being bound by any particular theory, codon optimization of the nucleotide sequence is considered to improve the translation efficiency of mRNA transcripts. Codon optimization of the nucleotide sequence can involve replacing a natural codon with another codon encoding the same amino acid but which can be translated by tRNA that is more readily available within the cell, thereby improving translation efficiency. nucleotide sequence optimization can also reduce secondary mRNA structures that interfere with translation, thus improving translation efficiency.

[0203] In one embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding any CAR described herein (including its functional portion and functional variants).

[0204] As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from a natural source (isolated and / or purified), may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, rather than phosphodiester linkages present between nucleotides of an unmodified oligonucleotide. In some embodiments, nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in some cases it may be suitable for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions.

[0205] Recombinant nucleic acids can be nucleic acids having sequences that are not naturally occurring or sequences prepared by artificial combination of two sequence segments (which would otherwise be separate). Such artificial combination is typically accomplished through chemical synthesis, or more commonly through artificial manipulation of isolated nucleic acid segments, such as through genetic engineering techniques, as described by Sambrook et al. (ibid.). Nucleic acids can be constructed using methods known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, for example, Sambrook et al. (ibid.) and Ausubel et al. (ibid.). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or a variety of modified nucleotides designed to improve the biological stability of the molecule or the physical stability of the resulting double strand (e.g., phosphate thioester derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, and 7-methylguanine. 5-Methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0206] Nucleic acids may contain any isolated or purified nucleotide sequence encoding any CAR or its functional portion or variant thereof. Alternatively, the nucleotide sequence may contain nucleotide sequences degenerate into any sequence or combination of degenerate sequences.

[0207] The implementation scheme also provides isolated or purified nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes to the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0208] Nucleotide sequences hybridized under stringent conditions can hybridize under highly stringent conditions. "Highly stringent conditions" refers to nucleotide sequences that specifically hybridize with the target sequence (the nucleotide sequence of any nucleic acid described herein), to a degree more detectable than non-specific hybridization. Highly stringent conditions include conditions that distinguish polynucleotides with precisely complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Such complementary small regions are easier to unwind than full-length complementary sequences of 14 to 17 or more bases, and highly stringent hybridization makes them easily distinguishable. Relatively highly stringent conditions would include, for example, low-salt and / or high-temperature conditions, provided, for example, by using about 0.02 to 0.1 M NaCl or equivalents at a temperature of about 50 to 70 °C. Such highly stringent conditions allow for small (if any) mismatches between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any of the CARs of this invention. It is generally recognized that conditions can be made even more stringent by adding an increased amount of formamide.

[0209] Also provided are nucleic acids containing nucleotide sequences having at least about 70% or more, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, identity with any nucleic acid described herein.

[0210] In one implementation, nucleic acids may be incorporated into a recombinant expression vector. At this point, the implementation provides a recombinant expression vector containing any nucleic acid. For the purposes of this document, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, a protein, a polypeptide, or a peptide and is brought into contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed intracellularly, allows the host cell to express the mRNA, protein, polypeptide, or peptide. The vector, as a whole, is not naturally occurring.

[0211] However, the vector portion can be naturally occurring. Recombinant expression vectors can contain any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially derived from natural sources, and can contain natural, non-natural, or altered nucleotides. Recombinant expression vectors can contain naturally occurring or non-natural nucleotide linkages, or both. Preferably, non-natural or altered nucleotides or nucleotide linkages do not impede transcription or replication of the vector.

[0212] In one implementation, the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. Vectors may be selected from the pUC family (Fermentas Life Sciences, GlenBurnie, MD), pBluescript family (Stratagene, LaJolla, CA), pET family (Novagen, Madison, WI), pGEX family (Pharmacia Biotech, Uppsala, Sweden), and pEX family (Clontech, Palo Alto, CA).

[0213] Phage vectors, such as λΰΤΙΟ, λϋΤΙ 1, λZapII (Stratagene), EMBL4, and λNMI 149, can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors can be viral vectors, such as retroviral vectors or lentiviral vectors. Lentiviral vectors are vectors derived from at least a portion of a lentiviral genome, particularly including self-inactivated lentiviral vectors, as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, LENTIVECTOR.RTM gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0214] Many transfection techniques are generally known in the field (see, for example, Graham et al., Virology, 52: 456-467 (1973); Sambrook et al. (ibid.); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13: 97 (1981).

[0215] Transfection methods include calcium phosphate coprecipitation (see, for example, Graham et al., ibid.), direct microinjection into cultured cells (see, for example, Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, for example, Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome-mediated gene transfer (see, for example, Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid-mediated transduction (see, for example, Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic acid delivery using high-speed microparticles (see, for example, Klein et al., Nature, 327: 70-73 (1987)).

[0216] In one implementation, recombinant expression vectors can be prepared using standard recombinant DNA techniques described below: for example, Sambrook et al., ibid., and Ausubel et al., ibid. Circular or linear expression vector constructs can be prepared to be contained within a functional replication system in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2 μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0217] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation start and stop codons, which, where appropriate, are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) to which the vector will be introduced, taking into account whether the vector is DNA- or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0218] Recombinant expression vectors may include one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes may contain biocidal resistance, such as resistance to antibiotics, heavy metals, etc., or be complementary in auxotrophic hosts to provide protrophic responses, etc. Suitable marker genes for use in the expression vectors of this invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0219] Recombinant expression vectors may contain natural or non-natural promoters operatively linked to a nucleotide sequence encoding a CAR (including its functional portion and functional variants) or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific, and developmentally specific) is within the general skill of those skilled in the art. Similarly, combinations of nucleotide sequences with promoters are also within the skill of those skilled in the art. Promoters may be non-viral or viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, or promoters found in long terminal repeats of mouse stem cell viruses.

[0220] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, they can be used for constitutive or inducible expression.

[0221] Furthermore, recombinant expression vectors can contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cell death in cells expressing a suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent (e.g., a drug) to cells expressing the gene, and causes cell death when the cell is exposed to or exposed to the reagent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase, and nitroreductase.

[0222] One embodiment also provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain the recombinant expression vectors of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α *Escherichia coli* (E. coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For amplification or replication of the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5α cell. For the production of a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0223] For the purposes of this article, a T cell can be any T cell, such as cultured T cells, like primary T cells, or T cells derived from cultured T cell lines (e.g., Jurkat, SupTl, etc.), or T cells derived from mammals. If derived from mammals, the T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. T cells can be CD8+ T cells or CD4+ T cells.

[0224] In one implementation, the CAR described herein can be used with suitable non-T cells. Such cells are cells with immune effector functions, such as NK cells and T-like cells derived from pluripotent stem cells.

[0225] One embodiment also provides a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population comprising host cells containing any of the recombinant expression vectors described herein, and additionally comprising at least one other cell type (e.g., a host cell, such as a T cell) that does not contain any recombinant expression vector, or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, myocytes, brain cells, etc. Alternatively, the cell population can be a substantially homogeneous population, wherein the population primarily comprises (e.g., substantially consists of) host cells containing the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell containing the recombinant expression vector, such that all cells in the population contain the recombinant expression vector. In one embodiment of the invention, the cell population is a clonal population comprising host cells containing the recombinant expression vector described herein.

[0226] CARs (including their functional portions and variants), nucleic acids, recombinant expression vectors, host cells (including their populations), and antibodies (including their antigen-binding portions) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are of higher purity than cells in their natural in vivo environment. Such host cells can be produced, for example, using standard purification techniques. In some embodiments, the host cell preparation is purified such that the host cells constitute at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, or about 80%, or can be about 100%.

[0227] E. Treatment methods

[0228] CARs intended for use in the treatment or prevention of mammalian diseases are intended for use in methods of utilizing active patient-specific autologous anti-tumor T cell populations. In this regard, one embodiment provides a method for treating or preventing cancer in mammals, comprising administering to the mammal an amount of CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or its antigen-binding moiety, and / or pharmaceutical composition effective in treating or preventing cancer in mammals.

[0229] One implementation also includes lymphodepleting of the mammalian lymphocytes prior to the administration of the CAR disclosed herein. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepletion chemotherapy, myeloablative lymphodepletion chemotherapy, and whole-body irradiation.

[0230] For the purposes of methods of administering host cells or cell populations, the cells can be mammalian allogeneic or autologous cells. Preferably, the cells are mammalian autologous. Allogeneic, as used herein, refers to any material derived from a different animal of the same species as the individual into which the material was introduced. Two or more individuals are considered allogeneic when the genes at one or more loci are not identical. In some respects, allogeneic material from individuals of the same species can be genetically sufficiently different to interact antigenically. "Autologous," as used herein, refers to any material derived from the same individual into which the material is subsequently reintroduced.

[0231] The term "mammal" as used herein refers to any mammal, including but not limited to rodents such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals may be from the order Carnivora, including felines (cats) and canines (dogs). Mammals may be from the order Artiodactyla, including bovids (cattle) and suidae (pigs), or include perissodactyls, including equines (horses). Mammals may be primates, ceboids, or simoids (monkeys), or anthropoids (humans and apes). Preferably, the mammal is a human.

[0232] For the method, cancer can be any cancer, including any of the following: acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma. Gastrointestinal carcinoid tumors, head and neck cancers (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, fluid-filled tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin lymphoma, B chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0233] The terms “treatment” and “prevention” as used herein, and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, which may be deemed by a person skilled in the art to have potential benefit or therapeutic effect. In this respect, the methods described can provide treatment or prevention of any amount or level of cancer in mammals.

[0234] Furthermore, the treatment or prevention provided by this method may include treating or preventing one or more conditions or symptoms of the disease being treated or prevented (e.g., cancer). Moreover, for the purposes of this article, "prevention" may encompass delaying the onset of the disease or its symptoms or conditions.

[0235] Another implementation provides a method for detecting the presence of cancer in mammals, comprising: (a) contacting a sample from a mammal containing one or more cells with a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or its antigen-binding portion or pharmaceutical composition to form a complex, and (b) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0236] Samples can be obtained by any suitable method, such as biopsy or necropsy. A biopsy involves removing tissue and / or cells from an individual. This removal can be the collection of tissue and / or cells from the individual for testing on the removed tissue and / or cells. The testing may include experiments to determine whether the individual has and / or suffers from a certain condition or disease state. A condition or disease may be, for example, cancer.

[0237] In an embodiment of a method for detecting the presence of proliferative disorders (such as cancer) in mammals, a sample containing mammalian cells may be a sample containing whole cells, their lysates, or fractions of whole-cell lysates (e.g., nucleus or cytoplasm fractions, total protein fractions, or nucleic acid fractions). If the sample contains whole cells, the cells may be any cells of a mammal, such as cells of any organ or tissue, including blood cells or endothelial cells.

[0238] Contact can occur in or outside the mammal. Preferably, the contact is external.

[0239] Furthermore, the detection of the complex can be performed in many ways known in the art. For example, the CARs disclosed herein, the peptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or their antigen-binding moieties can be labeled with detectable markers such as the radioisotopes disclosed above, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0240] Methods for testing the ability of CARs to recognize target cells and antigen specificity are known in the art. For example, Clay et al., J. Immunol, 163: 507-513 (1999) taught methods for measuring the release of cytokines such as interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), or interleukin-2 (IL-2). Furthermore, CAR function can be evaluated by measuring cellular cytotoxicity, as described by Zhao et al., J. Immunol. 174:4415-4423 (2005).

[0241] Another embodiment provides the use of the CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody or antigen-binding portion thereof, and / or pharmaceutical composition of the present invention for the treatment or prevention of proliferative diseases such as cancer in mammals. Cancer can be any cancer described herein.

[0242] Any method of administration may be used for the disclosed therapeutic agents, including local and systemic administration. For example, administration may be topically, orally, intravascularly (e.g., intravenously), intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, and subcutaneously. The specific administration route and dosage regimen are selected by the attending physician considering the details of the case (e.g., the patient, the disease, the disease state involved, and whether the treatment is prophylactic). In cases where more than one agent or composition is being administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, while antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. Administration methods include injection, wherein the CAR, CAR T cells, conjugates, antibodies, antigen-binding fragments, or compositions are provided in a non-toxic, pharmaceutically acceptable carrier such as water, saline, Ringer's solution, glucose solution, 5% human serum albumin, fixative oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying antibodies or antigen-binding fragments to areas of tissue removed from the tumor or areas suspected of being prone to tumor formation. In some implementations, sustained intratumoral (or peritumoral) release of a pharmaceutical formulation containing a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other instances, the conjugate is applied topically to the cornea as eye drops or administered intraocularly via the vitreous humor.

[0243] The disclosed therapeutic agent can be formulated into a single-dose unit suitable for precise dosage. Furthermore, the disclosed therapeutic agent can be administered in single-dose or multiple-dose regimens. A multiple-dose regimen is one in which the main treatment process may involve more than one separate dose, such as 1-10 doses, followed by additional doses at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment can involve daily or multiple-day doses of the compound over days to months or even years. Therefore, the dosage regimen will also be determined at least in part based on the specific needs of the subject being treated and will depend on the judgment of the administering physician.

[0244] Typical doses of antibodies or conjugates can be from about 0.01 to about 30 mg / kg, for example from about 0.1 to about 10 mg / kg.

[0245] In certain instances, a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR, CAR T-cell, or additional agent is administered to a subject over multiple daily dosing regimens, such as at least two consecutive days, 10 consecutive days, or over a period of time such as weeks, months, or years. In one instance, the conjugate, antibody, composition, or additional agent is administered to a subject for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0246] In some embodiments, the disclosed methods include combining the disclosed antibody, antigen-binding fragment, conjugate, CAR, or CAR-expressing T cells to deliver surgical, radiotherapy, and / or chemotherapy to a subject (e.g., sequentially, substantially simultaneously, or concurrently). The methods and dosages of these agents and treatments are known to those skilled in the art and can be determined by a skilled clinician. Preparation and administration regimens of additional agents may be used according to the manufacturer's instructions or determined empirically by a skilled practitioner. Preparation and administration regimens of such chemotherapy are also described in Chemotherapy Service, (1992) Ed., MC Perry, Williams & Wilkins, Baltimore, Md.

[0247] In some implementations, combination therapy may include administering a therapeutically effective amount of an additional cancer inhibitor to the subject. Non-limiting examples of other therapeutic agents that may be used with combination therapy include microtubule binders, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in a therapeutically effective amount) and the treatment may be used alone or in combination. For example, any suitable anticancer or antiangiogenic agent may be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. The methods and therapeutic dosages of these agents are known to those skilled in the art and can be determined by a skilled clinician.

[0248] Additional chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustard (e.g., chlorambucil, dichloroethylmethylamine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, formustine, lomustine, and streptozotocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, dichloromethyldiethylamine, procarbazine, temozolomide, thiotepa, and uramustine); antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; and plant-based chemotherapeutic agents. Alkaloids, such as podophyllotoxin (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vincristine, vinblastine, vinorelbine, and vinorelbine); cytotoxic / antitumor antibiotics, such as anthracycline members (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and pentorubicin), bleomycin, rifampin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtruzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, and porfimer. Sodium) and verteporfen; as well as other agents such as retinoic acid, hexamethylmelamine, azithromycin, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotinib, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estradiol, gefitinib, hydroxyurea, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotan, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and retinoic acid. The selection and therapeutic dosage of these agents are known to those skilled in the art and can be determined by a skilled clinician.

[0249] In some embodiments of the invention, cells activated and expanded (wherein T cells are expanded to therapeutic levels) using the methods described herein or other methods known in the art are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, during, or after them), including but not limited to treatments using pharmaceutical agents, such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natamizumab for MS patients, or efazolinumab for psoriasis patients, or other treatments for PML patients. In other embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immune scavengers such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines, and radiation. These drugs inhibit calcium-dependent phosphatases such as calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993). In other embodiments, the cell composition of the present invention is administered to a patient in combination with (e.g., before, during, or after) bone marrow transplantation, T-cell ablation therapy using a chemotherapy agent (such as fludarabine), external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B-cell ablation therapy such as rituximab, which reacts with CD20. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the present invention after transplantation. In other embodiments, the expanded cells are administered before or after surgery.

[0250] The amount of the above-mentioned therapeutic agent to be administered to the patient will vary depending on the condition being treated and the exact nature of the recipient. Dosage for human administration may be scaled according to practice accepted in the art. For example, for adult patients, the dose of CAMPATH is typically from 1 to approximately 100 mg, usually administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg, but in some cases larger doses of up to 40 mg per day may be used.

[0251] Combination therapy can provide synergistic effects and is demonstrated to be synergistic, meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the individual compounds. Synergistic effects are achieved when the active ingredients are co-formulated and administered or delivered simultaneously in unit dosage forms of the combination (2) as independent, alternating or parallel delivery; or (3) by other means. Synergistic effects are achieved when delivered alternately, for example by sequential administration or delivery through different injections in separate syringes. Generally, during alternation, the effective dose of each active ingredient is administered sequentially (i.e., continuously), while in combination therapy, the effective doses of two or more active ingredients are administered together.

[0252] In one implementation, following anticancer treatment, an effective amount of an antibody or antigen-binding fragment or conjugate specifically binding to one or more antigens disclosed herein is administered to a subject with a tumor. After sufficient time has elapsed to allow the administration of the antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, the immune complexes are detected. The presence (or absence) of the immune complexes indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained prior to treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control obtained prior to treatment indicates that the treatment is effective.

[0253] F. Biological drug compositions

[0254] This document provides biopharmaceutical or biological product compositions (hereinafter “Compositions”) for gene therapy, immunotherapy, adoptive immunotherapy, and / or cell therapy, said compositions comprising, for example, the disclosed CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier). The compositions may be formulated into unit dosage forms for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. The compositions may be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments are used, for example, to treat and detect tumors, such as, but not limited to, neuroblastoma. In some examples, the compositions may be used to treat or detect cancer. Compositions containing the CAR disclosed herein, or T cells expressing CAR, conjugates, antibodies, or antigen-binding fragments, are also used, for example, to detect pathological angiogenesis.

[0255] Compositions for administration may comprise a solution of a CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. Various aqueous carriers may be used, such as buffered saline solutions. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized using conventional, known sterilization techniques. The compositions may, as needed, contain pharmaceutically acceptable adjuvants to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these formulations, the concentration of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates, can vary widely and will be selected based on fluid volume, viscosity, body weight, etc., depending on the specific route of administration and the needs of the recipient. Practical methods for preparing such dosage forms for gene therapy, immunotherapy, and / or cell therapy are known or apparent to those skilled in the art.

[0256] Typical compositions for intravenous administration contain approximately 0.01 to approximately 30 mg / kg of antibody or antigen-binding fragment or conjugate per subject per day (or a corresponding dose of CAR or CAR-expressing T cells containing an antibody or antigen-binding fragment conjugate). Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0257] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water prior to administration, but they can also be provided as sterile solutions of known concentrations. The solution of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates, is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dose of 0.5 to 15 mg / kg body weight. There is considerable experience available in the art in administering antibody or antigen-binding fragment or conjugate drugs; for example, antibody drugs have been marketed in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, or their antigen-binding fragments or conjugates, can be administered by slow infusion rather than by intravenous bolus or rapid concentration. In one instance, a higher loading dose is administered, followed by maintenance at a lower dose. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (the corresponding dose of a conjugate containing antibody or antigen-binding fragment) can be infused over approximately 90 minutes, followed by a maintenance dose of 2 mg / kg per week infused over approximately 30 minutes for 4 to 9 weeks, provided the previous dose has been well tolerated.

[0258] Controlled-release parenteral formulations can be prepared as implants, oily injectables, or microparticle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Microparticle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein such as a cytotoxin or drug as a core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, so nanoparticles are only administered intravenously. Microparticles have a diameter of about 100 μm and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992).

[0259] Polymers can be used for the ion-controlled release of CARs, or CAR-expressing T cells, antibody or antigen-binding fragments or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous flowing liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective carrier for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). On the other hand, liposomes are used for the controlled release and targeting of drugs encapsulated in lipids (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous other systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0260] G. Reagent kit

[0261] In one aspect, kits for using the CARs disclosed herein are also provided. For example, kits for treating tumors in subjects or for preparing CAR T cells expressing one or more CARs disclosed herein. Kits typically contain the antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells may be included in the kit.

[0262] The kit may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers typically contain a composition comprising one or more of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells. In several embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The label or packaging insert indicates that the composition is intended to treat a specific condition.

[0263] Labels or packaging inserts will typically also include instructions for the use of disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells, for example, in methods of treating or preventing tumors or preparing CAR T cells. Packaging inserts often include instructions typically found in the commercial packaging of therapeutic products, containing information on indications, usage, dosage, administration, contraindications, and / or warnings for the use of such therapeutic products. Instructional materials may be written in electronic form (e.g., on a computer floppy disk or CD) or visual (e.g., video files). Kits may also contain additional components to facilitate the specific application for which the kit is designed. Thus, for example, a kit may additionally include means for detecting markers (e.g., enzyme-labeled enzyme substrates, filters for detecting fluorescent markers, appropriate secondary markers such as second antibodies, etc.). Kits may also include buffers and other reagents conventionally used for practicing specific methods. Such kits and suitable contents are well known to those skilled in the art.

[0264] Example

[0265] The invention is further illustrated by the following embodiments, which are not to be construed as limiting the scope of the invention in any way. Rather, it should be clearly understood that a variety of other embodiments, modifications, and equivalents may be made without departing from the spirit of the invention and / or the scope of the appended claims, as will be apparent to those skilled in the art upon reading the description herein.

[0266] The invention is further illustrated by the following embodiments, which are not to be construed as limiting the scope of the invention in any way. Rather, it should be clearly understood that a variety of other embodiments, modifications, and equivalents may be made without departing from the spirit of the invention and / or the scope of the appended claims, as will be apparent to those skilled in the art upon reading the description herein.

[0267] Example 1. Next-generation sequencing of tumor mutant genomes

[0268] This procedure involves next-generation sequencing of patient tumor material and identification of mutated proteins present in the tumor (as a group, referred to as the mutant group). These sequences will be used as the basis for generating vectors expressing mutated tumor proteins. When available, non-tumor-related patient material will be used for normal comparisons (e.g., peripheral blood), such as publicly available databases of the human genome. The next-generation sequencing method is a well-established technique in molecular biology and can be found, for example, in Vogelstein B, Papadopoulos N, Velculescu VE, et al., 2013, Cancer Genome Landscapes, Science 339:1546-1558.

[0269] The National Institutes of Health (NIH) provides the Cancer Genome Atlas online (cancergenome.nih.gov), which offers comprehensive maps of key genomic changes for 33 cancers. Data is delivered to the NIH through specific TCGA (The Cancer Genome Atlas) Genome Sequencing Centers (GSCs). For each TCGA cancer case sequenced, both whole-exome and whole-genome data are available. Non-tumor DNA is used as a control for each submission. Three centers funded by the National Human Genome Research Institute (NHGRI) provide whole-genome sequences: the Broad Institute Sequencing Platform (BroadInstitute, Cambridge, Mass.); the Human Genome Sequencing Center (Baylor College of Medicine, Houston, Texas); and the Genome Institute at Washington University, Washington University School of Medicine, St. Louis, Mo.

[0270] If someone signs up separately with the Broad Institute, the Human WES Express (Deep) service offers tumor-normal pair or somatic mutation analysis with 50× or higher coverage of 85% of the target bases (access information from June 10, 2016). www.genomics.broadinstitute.org / products / while-exome-sequencing Tumor samples can also be sequenced at Broad's CLIA-licensed CAP lab. Commercial whole-genome and whole-exome sequencing services are provided by Illumina. www.illumina.com / areas-of-interest / cancer / research.htlmlThe company now also offers a tumor immunogenicity discovery platform (ngs-immuno-oncology-application-spotlight-1170-2016-005-1.pdf). Other commercial providers are also available. This information is provided to demonstrate the widespread availability of whole-genome and whole-exome sequencing services in the market, and historically, the cost and speed of providing these sequences will continue to decline. Genomic analysis of human tumor samples is an easily accessible service, or can be performed in a laboratory using commercially available instruments and systems.

[0271] Example 2. Next-generation sequencing of TCR

[0272] This procedure involves using sequencing technology to define the fully complementary sequences of T-cell receptors in biological samples. The materials analyzed include patient tumors, in which case we will describe the TCRs present in the tumor. In peripheral blood, we will describe the common TCRs present, some of which are tumor-specific. Next-generation sequencing allows for the quantification of the frequency of specific TCRs. Applying next-generation sequencing to identify specific TCR α and β chain pairs is a well-established technique in molecular biology (Dash P, Wang G, Thomas P, 2015, Single-cell analysis of T-cell receptor ABrepertoire, in Immunosenecense: Methods and Protocols, Shaw AC (ed.), Methods in Molecular Biology, Vol. 1343, Springer Science+Business Media, New York).

[0273] Numerous methods have been developed using current molecular biology techniques, including the ongoing advancement of automated DNA sequencing, to determine the DNA sequences encoding the TCR α chain (TCRA) and TCR β chain (TCRB). Furthermore, many techniques have been developed to specify which TCRAs pair with TCRBs within the same T lymphocyte or within a population of T cells derived from clonal precursors. For example, in 2012, Sun et al. demonstrated the ability to sequence the TCR α and β chains at the single-cell level from phenotype-sorted CD8 T cells. (Sun X, Saito M, Sato Y, et al., 2012, Unbiased analysis of TCRA / B chains at the single-cell level in human CD8+ T-Cell subsets, PLoS ONE 7: e40386).

[0274] In 2014, Han et al. confirmed the sequencing of TCRA and TCRB from T cells, which are classified in certain cases by their ability to secrete specific subsets of cytokines, isolated using the Miltenyi Biotec cytokine capture system (immunomagnetic particles). (Han A, Gianville J, Hansmann L, Davis MM, 2014, Linking T-cell receptor sequence to functional phenotype at the single-cell level, Nature Biotechnology 32:684-692).

[0275] Similarly, sequences encoding the heavy and light chains of antibody libraries encoded by B cells have been analyzed using single-cell sequencing methods. (DeKosky B, Kojima T, Rodin A, et al., 2015, In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire, Nature Medicine 21:86-91.)

[0276] Example 3. Generation of lentiviral vectors expressing tumor mutant groups

[0277] To confer expression on a mutant group of antigen-presenting cells—antigen-presenting cells derived from a patient (a non-restrictive example being dendritic cells)—a lentiviral vector (LV) is used to encode ten of the most prevalent mutant proteins present in the mutant group (ten is an approximation, and the number of LVs can range from 1 to 100). The LV can encode the mutant group containing the relevant epitope, or each mutant gene can be cloned individually into multiple LVs. DCs can also be transduced with other genes or non-coding RNAs to enhance the production of highly functional DCs and / or T cells. Non-restrictive examples of such genes or non-coding RNAs are IL-2, IL-4, IL-12, IL-17, IL-15, IL-21, IL-7, IL-4, GM-CSF, miR21, miR221, and miR142-T. These proteins are also transduced to promote monocyte differentiation into DCs, and then, once differentiation occurs, are shut down using tissue-specific promoters and / or tissue-specific miRNAs known in the art.

[0278] LVs are rapidly produced by transducing production cell lines using a set of plasmids encoding the constituent genes required to produce the genetic vector. As required by current regulations, these plasmids are transfected into the production cell lines as a set. One of the transfected plasmids encodes the genetic payload of the LV, i.e., the desired gene to be delivered to the target cell line. Therefore, once the tumor mutant set is defined and the mutant genes expressed in antigen-presenting cells are selected, these genes are transferred into plasmids encoding one or more mutant proteins. LVs can hold up to 10,000 base pairs. Therefore, up to ten genes or individual genes can be encoded by an LV. If the allowed packaging gene size is exceeded, two or more LV populations are generated to encode the desired entire mutant set. The mutant genes encoding the mutant set are amplified by PCR or synthesized directly, with suitable sequences including those that allow for rapid cloning into the LV backbone plasmid (the plasmid encoding the target gene). Once LVs encoding the genes of the desired mutant set are generated, they are used to transduce antigen-presenting cells.

[0279] Example 4. Generation of lentiviral vectors expressing TCR

[0280] To confer expression of TCR sequences identified through sequencing of patient material, LV was used to encode the full-length TCRA and TCRB chains. These vectors were then used to transduce patient T cells, thereby generating multispecific T cells (both native and transduced TCRs).

[0281] The ability to molecularly clone, sequence, and transfer human TCRs into primary human T cells using retroviral gene vectors is well-established in this field. Transduced T cells acquire the ability to target cells using the vector-transferred TCR. If the transduced T cells are cloned, it can be demonstrated that both the native and transduced TCRs are functional (Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encodedpeptide, Clinical Immunology, 98:220-228; see also Jurgens et al., 2006, Transduction of primary lymphocytes with Epstein-Barr virus (EBV) latentmembrane protein-specific T-cell receptor induces lysis of virus-infected cells: a novel strategy for the treatment of Hodgkin's disease and nasopharyngeal carcinoma, J Clinical Immunology, 26:22-32).

[0282] LVs encoding single or multiple TCRs, for example, whose TCRA and TCRB sequences are derived from T cells isolated from ovarian cancer patients, are generated; and these LVs are used to transduce autologous patient lymphocytes that have been isolated, activated, and cultured in vitro. Examples of culture media used include RPMI-1640 or TexMACS, supplemented with or without human serum or human serum albumin, and supporting cytokines such as IL-2, IL-7, IL-15, IL-21, or combinations thereof. Activation is facilitated by using nanomatrices with anti-CD3 and anti-CD28 binding properties, such as the Mitenyi TransACT system. Culture is performed according to standard techniques in the art (i.e., in tissue culture flasks) or on automated culture platforms (e.g., CliniMACS Prodigy (Miltenyi Biotec)). The presence of novel TCRs on the surface of the transduced T cell population from the patient can be confirmed by antibody staining for the transduced specific TCRBs or by PCR using these sequences.

[0283] The activated T cell population (now containing TCRs cloned from the patient) is then used to recognize the cancer antigens expressed by the patient. For example, if the TCRs were originally cloned from patient-derived T cells activated by dendritic cells expressing antigen X, the transduced T cell population is now activated after co-culturing with APCs (e.g., dendritic cells or B cells) that have been transduced or transfected to express antigen X. Antitumor activity was demonstrated after this T cell population was transferred into the patient.

[0284] In another example, LV-encoded inhibitors of natural TCRs, such as antisense or shRNAs that specifically target endogenous TCRs but not the TCR encoded in the vector, wherein the encoded TCR is modified to resist the action of the antisense or shRNA, thereby generating tumor-specific T cells that target the antigen but not the endogenous TCR. These engineered T cells may have improved safety and efficacy properties compared to T cells that also express endogenous TCRs.

[0285] In this embodiment, LVs expressing both TCR and CAR were generated to achieve anti-tumor effects. TCR and CAR can be expressed on the same vector or different vectors. A preferred embodiment is to generate multiple vectors to express the desired CAR, TCR, and any other gene or non-coding nucleic acid (collectively, payloads) that can enhance the therapeutic or preventative effects of the pharmaceutical product.

[0286] Example 5. Generation of lentiviral vectors expressing CAR

[0287] A key element is transducing the patient's T cells with a chimeric antigen receptor (CAR). The CAR must be expressed at a sufficient level on the surface of the T cells to ensure activation of the transduced T cells upon encountering a CAR target cell. For example, this can be achieved by stimulating T cells carrying a CD19 CAR with normal B cells expressing CD19 or with CD19-expressing leukemia cells. The CAR is not specific to the identified mutant tumor protein but rather targets normal B cells or other depleting cell types that may be present in the tumor microenvironment, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), tumor-associated fibroblasts or fibroblasts, or other cell types present in the tumor stroma.

[0288] In the case of B cells, the safety profile of CD19 and CD20-specific CARs has been well established. Dual CARs targeting both CD19 and CD20 can also be used. These are responsive to these heterologous or autoantigens, which will drive the expansion of tumor-specific T cells after in vivo infusion or perhaps even in vitro during culture (e.g., if the antigen is shared with dendritic cells). A non-exhaustive list of antigens is as follows: CD19, CD20, CD22, CD33, CD38, CD14, CD11b, TIE-2, VEGFR1, VEGFR2. As a non-exhaustive example, or as described above, DCs can be further modified to enhance activity by expressing, for example, GM-CSF, IL-4, TRP2, and / or IFN-α. DCs can also be infused into patients if desired. In this way, DCs will be used to induce or enhance the activity of transduced T cell populations that now express homologous TCRs in vivo.

[0289] A non-limiting example is the inclusion of elements within the vector that allow for better fine-tuning of load expression to enhance or optimize the desired effect. These include, but are not limited to, genetic switches, suicide genes, and rheostat elements. For instance, CAR expression may only be desired for a period of time after treatment, and it may be preferable to turn off CAR expression in vivo while maintaining TCR expression long-term, allowing the modified T cells to continuously detect tumor cells in vivo.

[0290] Example 6. Culturing DCs and transducing them with lentiviral vectors expressing mutant genome libraries (DCmutn)

[0291] To present mutant proteins to patient T cells, autologous antigen-presenting cells, such as dendritic cells (DCs), are transduced to express the mutant protein encoded by the mutant group, with the specific protein expressed defined by the mutant protein most highly expressed in the tumor. This in vitro procedure allows for precise analysis and evaluation of the immunotherapeutic T cell population prior to infusion.

[0292] One non-limiting example involves isolating monocytes from a patient's peripheral blood under non-GMP conditions and first transducing them with multiple lower venous cells (LVs) expressing the mutated antigen, followed by differentiation into dendritic cells using soluble IL-4 and GM-CSF. Once the cells have differentiated, patient T cells are passaged together with dendritic cells to expand tumor-specific T cells. Tumor-specific T cells are then isolated using various methods, and specific TCRs are sequenced and measured. These TCRs are then synthesized and cloned into LVs for use as a drug product in a vector prepared under GMP conditions.

[0293] Another non-limiting example uses the same mononuclear cell isolation and LV-mediated antigen-specific dendritic cell generation, but under GMP conditions. Patient T cells are transduced with LV-anti-CD19 CARs, then cultured with genetically modified DCs for less than 4 days, and then the antigen-specific T cells, along with possibly antigen-expressing DCs, are infused back into the patient as a therapeutic product.

[0294] Example 7. Transduction of patient PBMCs using CAR (T-CAR)

[0295] To promote T cell proliferation and evade tumor suppressor signals in vivo, patient T cells are transduced with CARs (such as CARs targeting CD19, CD20, or other depleting autoantigens). A CAR contains both "signal 1" and "signal 2." "Signal 1" is provided, for example, by the CR3 ζ chain (signal 1 refers to the signal normally activated by the TCR upon encountering the homopeptide-MHC complex and includes phosphorylation of the TCR-ζ chain). "Signal 2" is provided by CD137, CD28, or other T cell signaling molecules known to play a role in T cell activation and inducing T cell proliferation and persistence (signal 2 refers to those biologically required to allow T cells that have received signal 1 to be further stimulated and persisted in vitro or in vivo, and may include activation of the Jak-STAT pathway, PI3 kinase, PKC isoforms, TRAF pathway, or NF-κB pathway). Signal 1 and signal 2 may be encoded by the same CAR construct or may be distributed across gene products encoded by different LVs, which will activate T cells upon encountering a specific CAR ligand. As a means of ensuring the persistence of the patient's T-cell population transduced by TCR, the expression of CAR constructs is a central aspect of the adoptive immunotherapy described in this article, in which CARs provide persistence and survival signals for T cells, even when tumor-specific TCRs are insufficient to do so.

[0296] Example 8. Transduction of patient PBMCs (recT) using TCR

[0297] LVs expressing TCRA and TCRB chains identified by tumor and peripheral blood sequencing are generated. Depending on the number of identified TCRA and TCRB pairs, an LV may encode multiple TCRs, or generate multiple LVs with a single TCR, or a combination of both. Specific techniques for identifying TCRA and TCRB chain pairings are used in the design of these vectors, as described in detail above in the section on DNA sequencing-based TCR identification. These T cells are reactive to LV-transduced DCs or B cells or tumor mutant sets presented by in vivo tumor cells. Therefore, a TCR sequence from an ovarian cancer patient (from peripheral blood or from tumor resection lymphocytes, identified as tumor-reactive, for example by expressing a set of activation markers or by reactivity with APCs expressing tumor-encoded proteins (i.e., part of the mutant set) is cloned into an LV vector, and the patient's T cells are transduced with this vector, resulting in a transduced T cell population that now expresses a tumor-reactive TCR. The LV-transduced T cell population is tumor-reactive and can be re-infused into the patient.

[0298] Example 9. Transduction of patient PBMCs using CAR and TCR (recT-CAR)

[0299] In some cases, patient T cells are transduced with at least one CAR and multiple recombinant TCR sequences (recT). These engineered multispecific T cells are able to respond to tumor cells via native TCRs or recTs, enhancing anti-tumor effects, and the CARs sustain the T cell population. In this case, a population of T cells from a patient with ovarian cancer is transduced using an LV vector encoding both a TCR (originally derived from the patient and identified as tumor-reactive) and a CAR. The TCR is used to activate and direct anti-tumor activity, and the CAR is used to sustain the therapeutic T cell population in vivo. For a single-build scenario, the ovarian tumor is sequenced at the genomic or exon level, and tumor antigen X is identified. Antigen X is then transduced via LV to express in autologous APCs such as dendritic cells. Patient lymphocytes are then co-incubated with DCs expressing X, and the reactive cells are sequenced to identify TCRA and TCRB sequences. The TCRA and TCRB pairs from this sequencing are then used to construct an LV expressing an X-specific TCR. Alternatively, tumor antigen-reactive T cells are identified by other activation markers directly from blood or tumor tissue, the TCRA and TCRB sequences are identified, and cloned into the LV. Patient T cells are then activated in vitro using the TrasnAct reagent (which stimulates T cells via CD3 and CD28). The activated T cells are then transduced using either two independent LVs, one encoding a TCR and the second encoding a TCR responsive to X; or a single vector co-expressing CAR and TCR. The transduced T cell population is then expanded in culture to demonstrate transgene expression. Once LV-encoding sequence expression is confirmed, the therapeutic T cell population is infused back into the patient for anti-cancer effects. This approach can be achieved by increasing X to include a greater number of tumor-associated mutant proteins (mutant group products). Alternatively, this approach can be achieved by identifying more than one TCR associated with anti-tumor cells or by recognizing reactivity with APCs expressing multiple tumor antigens from the mutant group. The effector T cell population is then infused back into the patient for therapeutic effects, thus either a polyclonal T cell population expressing a single TCR specific to X and CAR, or a polyclonal T cell population expressing multiple TCRs responsive to multiple cancer antigens and co-expressing CAR. This key, inventive step describes a novel population of effector T cells derived from patients that has been engineered to express CARs targeting non-essential antigens encoded by normal tissues (such as CD19 or CD20), as well as tumor-specific TCRs.

[0300] Example 10. Co-culture of T cell population and transduced DCs

[0301] To expand tumor-reactive T cells (whether transduced with CAR, recT, or CAR-CAR), T cells are co-cultured with dendritic cells (DCs) expressing a subset of the tumor mutant group. In one implementation, recT-expressing cells do not need to be cultured on DCs, as the recT + CAR combination is sufficient to expand tumor-reactive T cells in vivo. Co-culturing with antigen-presenting cells such as DCs has confirmed the tumor reactivity of TCRA and TCRB expression vectors and can be performed as a routine assay. Cells are cultured with a variety of possible cytokines or other factors to enhance the generation or identification of antigen-specific T cells. APCs or DCs can also be cultured in the presence of factors to further enhance antigen-specific T cell expansion. Non-limiting examples include the addition of anti-PD1 inhibitors or IL-12, but many possible factors are tested and their enhancing effects evaluated during co-culture.

[0302] Example 11. Expanding the RecT-CAR-T population by co-administering or sequentially administering autologous cell products capable of providing CAR or RecT-mediated signaling to a therapeutic T cell population.

[0303] In one variation of this procedure, both LV-mutant transduced DCs (or other APCs) and effector T cell populations can be infused or injected into the patient. It is also conceivable that this second cell population could be cultured for a period before perfusion, or cryopreserved and administered at single or multiple consecutive times. For example, subcutaneous injection of DCs expressing the mutant group into lymph nodes or other sites in the body can enhance the expansion and function of intravenously injected recTs or natural antitumor TCRs. In this case, CAR expression amplification drives the transduced T cell population upon encountering a CAR-specific normal antigen. The introduction of a dendritic cell population expressing proteins encoded by the mutant group serves to drive antitumor T cell function via recT expression from the T cell population. It is also possible to eliminate the CAR-driving autoantigen (e.g., CD19) to the point where it no longer amplifies the therapeutic T cell population. In this case, autologous APCs (e.g., dendritic cells or cryopreserved B cells) or inactivated Epstein-Barr virus immortalized B cells can be used to expand the recT-CAR-T population. In addition, immortalized B cell lines can also be used to express mutant histones.

[0304] Immortification of patient B cells using EBV in academic laboratories (e.g., University of North Carolina School of Medicine, see...). https: / / unclineberger.org / research / core-facilities / tissueculture / b-cell-immortalization-services ) and as a commercial service (see, for example, AppliedBiologic Material, ABM, Inc., https: / / www.abmgood.com / EBV-Cell-Immortalization.html Standard services are available in both cases. Here, the patient's B cells are exposed to EBV in the form of culture supernatant, and transformed B cell colonies are amplified. These patient-derived autologous cells are commonly used in genetic, virological, and immunological procedures.

[0305] Therefore, this helper autologous cell product will be used to expand a therapeutic T cell population by expressing both the CAR target and the recT target antigen. If the helper APC product does not express the CAR target, it will stimulate the therapeutic T cell population by expressing mutant histones alone.

[0306] Example 12. Generating a specific T-cell population for immunotherapy

[0307] The compositions and methods described herein generate numerous T-cell populations suitable for adoptive immunotherapy. In all cases, when CARs are included, the aim is not to react with the tumor antigen itself, but to drive the expansion of patient T cells or target immunosuppressive cells, with or without recT co-expression. These cell populations can be summarized as follows:

[0308] A. T-CARs cultured with DCmutn, wherein the T-CARs target immunosuppressive cell targets, and DCmutn expands antigen-specific T cells.

[0309] B. recT-CARs not co-cultured with DCmutn cells, wherein recT-CARs are genetically modified T cells that also express CAR, but the cells themselves are not cultured on DCs. recT TCRs are identified by culturing independent groups of T cells co-cultured with DCmutn cells.

[0310] C. recT-CAR cultured with DCmutn, wherein rec-T CAR cells are generated by transducing patient T cells with CAR and culturing said cells on DCmutn cells to expand and isolate antigen-specific T cells that also express CAR targeting a population of tumor suppressor cells, and for long-lived / expanded T cell populations.

[0311] D. recT cells not co-cultured with DCmutn cells, wherein rec-T cells are T cells that do not express the genetically modified CAR and are not cultured on DCs themselves. rec-TTCRs were identified by culturing independent groups of T cells co-cultured with DCmutn cells.

[0312] E. recT cells cultured with DCmutn cells, wherein rec-T cells are cultured with DCmutn cells to obtain TCR antigen-specific T cells.

[0313] F. DC-mutant populations used in vitro and also as in vivo adjuvants / vaccines, wherein the DC-mutant populations are used as vaccines to drive the expansion of rec-T or rec-T CAR cells in the body.

[0314] Example 13. Alternative Donors and T Cell Types

[0315] Regarding alternative donors and T cell types, consider the two important variations of adoptive immunotherapy methods described in this article.

[0316] The first variation is adoptive immunotherapy in the context of hematopoietic stem cell transplantation (HSCT). HSCT has been attempted in both hematologic malignancies and solid tumors. To apply the procedure described here, post-HSCT, a population of dendritic cells (DCs) (or other APCs) and T cells is derived from a bone marrow (HSC) donor, and therapeutic T cells are generated and infused into the post-HSCT.

[0317] Therefore, patients with conditions such as myeloma have had their malignant tumors sequenced, and mutant groups identified. Mutant histone antigens are expressed in APCs derived from HSC donors (via LV transduction). HSC donor-derived T cells are activated and selected for direct use, or co-cultured with APCs expressing proteins encoded by the mutant group for TCR sequencing. Because of their reactivity to normal autoantigens, the CAR construct remains identical to the technology used in non-HSCT applications.

[0318] The second variation involves adoptive immunotherapy using alternative T cell populations. Cell surface activation markers such as CD137, CD69, PD-1, CD25, and class II MHC are well-known and frequently used to identify and isolate activated T cell populations (e.g., using Miltenyi Biotec CliniMACS CD137-Biotin reagent or CliniMACS CD25 reagent). Activated T cell populations are isolated from peripheral blood, tumors, or after exposure to dendritic cells (DCmutns) expressing tumor-associated antigens using these methods. Similarly, the ability to isolate activated T lymphocytes that produce activation-related cytokines can be used as a means of isolating tumor antigen-responsive T cells (e.g., using the Miltenyi Biotec CliniMACS cytokine capture system (IFN-γ)). Effector T cell populations are also sorted into specific cell populations using methods such as magnetic bead sorting, flow cytometry, solid-phase antibodies bound to plastic surfaces, and solid-bound ligands to desired markers expressed by the desired T cell type that is expected to adhere to the matrix, in a manner that defines the T cell type by the expression of cell surface proteins (markers). For example, CD4 or CD8 immunomagnetic beads (e.g., using Miltenyi Biotec CliniMACS CD4 reagent or CliniMACS CD8 reagent) can be used to isolate CD4 cells that respond to mutant peptides that bind to class II MHC or CD8 cells that respond to mutant peptides that bind to class I MHC. These cell types can then be used as a single population (e.g., CD4 only) or in specific combinations or ratios. Similarly, biomarkers for T cell differentiation have been used to select specific populations for adoptive immunotherapy. These differentiation biomarkers are used for positive or negative selection of memory T cell populations or naive T cell populations (e.g., using CliniMACS CD45RA reagent or CliniMACS CD62L reagent). In addition, specific physiological aspects of T cell populations can be used to identify more primitive T cell populations that can be better expanded in vivo (e.g., using reagents that identify cell populations expressing aldehyde dehydrogenase on the surface of T cells or expressing specific combinations of sodium (Na+) and potassium (K+) channels, see Liepins A, et al., 1989, “Serotonin modulated Ca++ dependent K+ channels in alloimmune effector cell lytic function. ImmunopharmacolImmunotoxicol 11:165-178, and Gallin EK, 1986, Ionic channels in leukocytes, J Leukoc Bio 39:241-254).Therefore, in the above embodiments, a subset of T cells from myeloma patients is isolated as a therapeutic cell population before culturing T cells from APCs (DCmutn) or after exposing an unselected T cell population to APCs but before infusion into the patient. In one application, these biomarkers or physiological characteristics are used to more accurately identify tumor-reactive T cells, thus serving as a basis for more effective identification of TCRA and TCRB sequences. In another application, a T cell population for immunotherapy is pre-selected for certain biomarkers before infusion into the patient but after recTCR and CAR induction expression via LV transduction. In yet another application, T cells expressing tumor-reactive biomarkers are selected after isolation from the patient, and this selected subset is co-cultured with APCs (DCmutn) to more effectively identify tumor-specific TCRA and RCRB sequences.

[0319] While numerous details have been described in conjunction with the exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and / or substantially equivalents will become apparent upon reading the foregoing disclosure, whether they are known or not currently foreseen.

[0320] Every application and patent cited herein, and every document and reference cited in each application and patent (including during the examination of each granted patent) (“Application References”), and every PCT and foreign application or patent corresponding to these applications and patents and / or claiming priority to any of these applications and patents, and every document cited or referenced in each Application References, is expressly incorporated herein by reference and may be used in the practice of this invention. More generally, documents or references are cited herein in the list of references preceding the claims or in the document itself; each of these documents or references (“References Cited herein”) and every document or reference cited in each References herein (including any manufacturer’s instructions, specifications, etc.) is expressly incorporated herein by reference.

[0321] The foregoing description of some specific embodiments provides sufficient information that others can readily modify or adapt these specific embodiments for various applications by applying current knowledge without departing from the general concept. Therefore, such modifications and adaptations should and are intended to be covered within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not for limiting purposes. Exemplary embodiments have been disclosed in the drawings and specification, and although specific terms may have been used, they are used only in a general and descriptive sense and not for limiting purposes, unless otherwise indicated, and therefore the scope of the claims is not limited thereto. Moreover, those skilled in the art will understand that certain steps of the methods discussed herein may be ordered in an alternative order, or that steps may be combined. Therefore, it is intended that the appended claims are not limited to the specific embodiments disclosed herein. Those skilled in the art will recognize, or be able to, determine many equivalents of the embodiments of the invention described herein using only conventional experimentation. The following claims cover such equivalents.

[0322] Reference sequence list

[0323] This application contains a sequence list to be electronically filed with the United States Patent and Trademark Receiving Office via a PDF file entitled “Sequence Listing”. This sequence list is incorporated by reference.

[0324] Sequence of this disclosure

[0325] The nucleic acid and amino acid sequences listed below use standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but it should be understood that the complementary strand, shown by reference, is also included. In the attached sequence listing:

[0326] SEQ ID NO:5 is the nucleotide sequence of the leader / signal peptide sequence:

[0327]

[0328] SEQ ID NO:6 is the amino acid sequence of the leader / signal peptide sequence:

[0329]

[0330] SEQ ID NO.:11 is the nucleotide sequence of the DNA CD8 transmembrane domain:

[0331]

[0332] SEQ ID NO:12 is the amino acid sequence of the CD8 transmembrane domain:

[0333]

[0334] SEQ ID NO:13 is the nucleotide sequence of the CD8 hinge domain of DNA:

[0335]

[0336] SEQ ID NO:14 is the amino acid sequence of the CD8 hinge domain:

[0337]

[0338] SEQ ID NO:15 is the amino acid sequence of amino acids 118 to 178 of the hinge region of CD8.α (NCBI RefSeq: NP.sub.--001759.3):

[0339]

[0340] SEQ ID NO:16 is the amino acid sequence of human IgG CL sequence:

[0341]

[0342]

[0343] SEQ ID NO 17 is the nucleotide sequence of the DNA signaling domain of 4-1BB:

[0344]

[0345] SEQ ID NO:18 is the amino acid sequence of the signal transduction domain of 4-1BB:

[0346]

[0347] SEQ ID NO:19 is the nucleotide sequence of the CD3-ζ DNA signaling domain:

[0348]

[0349] SEQ ID NO:20 is the amino acid sequence of CD3ζ:

[0350]

[0351]

[0352] SEQ ID NO:21 is the nucleotide sequence of the nucleic acid sequence (DNA) SP-CD19 conjugate-CD8 linker-CD4tm-signal LTG1562:

[0353]

[0354] SEQ ID NO:22 is the amino acid sequence of the SP-CD19 conjugate-CD8 linked-CD4tm-signal LTG1562:

[0355]

[0356] SEQ ID NO:27 is the nucleotide sequence of Scvf cd 19:

[0357]

[0358] SEQ ID NO:28 is the amino acid sequence of Scvf cd 19:

[0359]

[0360]

[0361] SEQ ID NO:29 is the nucleotide sequence of SP-CD19 conjugate-CD8 linker-CD8tm-signal transduction LTG1494 (see, Figure 3A, applicant’s co-pending provisional patent application number 62 / 239,509):

[0362]

[0363] SEQ ID NO:30 is the amino acid sequence of SP-CD19 conjugate-CD8 linker-CD8tm-signal transduction LTG1494 (see, Figure 3A, applicant's co-pending provisional patent application number 62 / 239,509):

[0364]

[0365] SEQ ID NO:31 is the nucleotide sequence of SP-CD19 conjugate-CD8 linker-CD8tm-signal (LTI reengineered) (LTG1538) (see Figure 3B of the applicant’s co-pending provisional patent application No. 62 / 239,509):

[0366]

[0367]

[0368] SEQ ID NO: 32 is the amino acid sequence of the SP-CD19 conjugate-CD8 linker-CD8tm-signal (LTI reengineered) (LTG1538) (see Figure 3B of the applicant’s co-pending provisional patent application No. 62 / 239,509):

[0369]

[0370] The technical solutions corresponding to the original claims of the parent application are hereby incorporated in this specification:

[0371] 1. An adoptive immunotherapy composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0372] 2. The adoptive immunotherapy composition of Item 1 further comprises an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors, wherein the T cell population is additionally transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population, the active patient-specific autologous anti-tumor T cell population being able to promote in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0373] 3. An adoptive immunotherapy composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors, wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0374] 4. The adoptive immunotherapy composition of item 2 or 3, wherein the tumor-specific T-cell receptor (TCR) is first identified by co-culturing antigen-presenting cells (APCs) transduced with one or more lentiviral vectors expressing patient-derived tumor antigens with HLA-compatible or patient-specific T cells.

[0375] 5. The adoptive immunotherapy composition of item 2 or 3, wherein the autoantigen-presenting cells are derived from autologous dendritic cells or B cells or a mixture thereof or peripheral blood-derived lymphocytes.

[0376] 6. The adoptive immunotherapy composition of item 2 or 3, wherein the tumor-specific T-cell receptor (TCR) is HLA-compatible or patient-specific.

[0377] 7. The adoptive immunotherapy composition of Item 1, wherein autologous patient-specific T cells containing a natural T cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active population of patient-specific autologous anti-tumor T cells, said active population of patient-specific autologous anti-tumor T cells being able to promote in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0378] 8. The adoptive immunotherapy composition of item 2 or 3, wherein the autologous patient-specific T cells containing a patient-specific, tumor-specific T cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active population of patient-specific autologous anti-tumor T cells, the active population of patient-specific autologous anti-tumor T cells being able to recognize the tumor-specific T cell receptor (TCR) and being able to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0379] 9. The adoptive immunotherapy composition of item 1 or 2, wherein the patient-derived tumor antigen is identified by patient biopsy and nucleotide sequencing to identify mutant RNA transcripts within the mutant group.

[0380] 10. The adoptive immunotherapy composition of item 1 or 2, wherein the autologous anti-tumor T cell population comprises autologous antigen-presenting cells (APCs), wherein the autologous antigen-presenting cells comprise patient-specific dendritic cells or B cells or a mixture thereof or peripheral blood-derived lymphocytes.

[0381] 11. The adoptive immunotherapy composition of item 2 or 3, wherein the CAR comprises at least one extracellular antigen-binding domain, at least one adapter domain, at least one transmembrane domain and at least one intracellular signal transduction domain.

[0382] 12. The adoptive immunotherapy composition of item 2 or 3, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one single-chain variable fragment of an antibody that binds to the antigen.

[0383] 13. The adoptive immunotherapy composition of item 2 or 3, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one heavy chain variable region of an antibody that binds to the antigen.

[0384] 14. The adoptive immunotherapy composition of item 2 or 3, wherein at least one extracellular antigen-binding domain of the CAR, at least one intracellular signal transduction domain of the CAR, or both are connected to a transmembrane domain via a linker or spacer subdomain.

[0385] 15. The adoptive immunotherapy composition of item 2 or 3, wherein the extracellular antigen-binding domain of the CAR is preceded by a lead peptide.

[0386] 16. The adoptive immunotherapy composition of item 2 or 3, wherein the extracellular antigen-binding domain of the CAR targets an antigen comprising CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR, or any combination thereof.

[0387] 17. The adoptive immunotherapy composition of item 2 or 3, wherein the extracellular antigen-binding domain of the CAR comprises an anti-CD19 scFV antigen-binding domain, an anti-CD20 scFV antigen-binding domain, an anti-CD22 scFV antigen-binding domain, an anti-ROR1 scFV antigen-binding domain, an anti-TSLPR scFV antigen-binding domain, an anti-mesothelin scFV antigen-binding domain, an anti-CD33 scFV antigen-binding domain, an anti-CD38 scFV antigen-binding domain, an anti-CD123 (IL3RA) scFV antigen-binding domain, an anti-CD138 scFV antigen-binding domain, an anti-BCMA (CD269) scFV antigen-binding domain, an anti-GPC2 scFV antigen-binding domain, an anti-GPC3 scFV antigen-binding domain, an anti-FGFR4 scFV antigen-binding domain, an anti-c-Met scFV antigen-binding domain, and an anti-PMSA domain. The scFV antigen-binding domain, the anti-glycolipid F77 scFV antigen-binding domain, the anti-EGFRvIII scFV antigen-binding domain, the anti-GD-2 scFV antigen-binding domain, the anti-NY-ESo-1TCR scFV antigen-binding domain, the anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0388] 18. The adoptive immunotherapy composition of item 2 or 3, wherein the CAR's linker or spacer domain is derived from the extracellular domain of CD8 and is connected to the transmembrane domain.

[0389] 19. The adoptive immunotherapy composition of item 2 or 3, wherein the CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from: the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, or any combination thereof.

[0390] 20. The adoptive immunotherapy composition of item 2 or 3, wherein the at least one intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0391] 21. The adoptive immunotherapy composition of item 2 or 3, wherein at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

[0392] 22. The adoptive immunotherapy composition of item 2 or 3, wherein the at least one intracellular signal transduction domain comprises a co-stimulatory domain, a primary signal transduction domain, or any combination thereof.

[0393] 23. The adoptive immunotherapy composition of item 2 or 3, wherein the at least one co-stimulatory domain comprises functional signal transduction domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or any combination thereof.

[0394] 24. A pharmaceutical composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0395] 25. The pharmaceutical composition of item 24 further comprises an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cell population is additionally transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of recognizing the tumor-specific T cell receptors (TCRs) and capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0396] 26. A pharmaceutical composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population, the active patient-specific autologous anti-tumor T cell population being capable of recognizing the tumor-specific T cell receptors (TCRs) and being capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0397] 27. The pharmaceutical composition of item 25 or 26, wherein the T cells are T cells of a person suffering from blood cancer.

[0398] 28. The pharmaceutical composition described in item 25 or 26, wherein the blood cancer is leukemia or lymphoma.

[0399] 29. The pharmaceutical composition described in item 25 or 26, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0400] 30. The pharmaceutical composition described in item 25 or 26, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

[0401] 31. The pharmaceutical composition described in item 25 or 26, wherein the blood cancer is multiple myeloma.

[0402] 32. The pharmaceutical composition described in item 25 or 26, wherein human cancers include adult epithelial cancers, including oropharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile ducts, gallbladder, pancreas), respiratory system cancers (larynx, lungs and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0403] 33. A method for treating mammals suffering from diseases, disorders, or conditions associated with elevated tumor antigen expression, the method comprising administering to a subject a pharmaceutical composition comprising an antitumor-effective amount of an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous antitumor T cell population capable of promoting in vivo expansion and persistence of patient-specific antitumor T cells in a patient-specific manner, leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer.

[0404] 34. The method of Item 33, further comprising an antitumor effective amount of an autologous T-cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T-cell population is additionally transduced with one or more lentiviral vectors encoding tumor-specific T-cell receptors (TCRs) to generate an active patient-specific autologous antitumor T-cell population capable of recognizing the tumor-specific T-cell receptors (TCRs), wherein the active patient-specific autologous antitumor T-cell population can be directly infused back into the patient to promote in vivo expansion and persistence of patient-specific antitumor T-cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

[0405] 35. A method for treating mammals suffering from diseases, disorders, or conditions associated with elevated tumor antigen expression, the method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising an antitumor-effective amount of an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous antitumor T cell population capable of recognizing the tumor-specific T cell receptors (TCRs), the active patient-specific autologous antitumor T cell population being capable of being directly infused back into the patient to promote in vivo expansion and persistence of patient-specific antitumor T cells in a patient-specific manner, leading to tumor stabilization, reduction, and / or elimination, and / or remission and / or elimination of cancer.

[0406] 36. The methods described in items 34 and 35, wherein the T cells have been pre-selected by expressing specific activation or memory-related surface markers.

[0407] 37. The methods described in items 34 and 35, wherein the T cells and dendritic cells are derived from a hematopoietic stem cell donor, and wherein the process is performed in the case of hematopoietic stem cell transplantation.

Claims

1. An adoptive immunotherapy composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (CARs), wherein the T cells are co-cultured with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

2. The adoptive immunotherapy composition of claim 1, further comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors, wherein the T cell population is additionally transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

3. An adoptive immunotherapy composition comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors, wherein the T cell population is further transduced with one or more lentiviral vectors encoding tumor-specific T cell receptors (TCRs) to generate an active patient-specific autologous anti-tumor T cell population capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

4. The adoptive immunotherapy composition of claim 2 or 3, wherein the tumor-specific T-cell receptor (TCR) is first identified by co-culturing antigen-presenting cells (APCs) transduced with one or more lentiviral vectors expressing patient-derived tumor antigens with HLA-compatible or patient-specific T cells.

5. The adoptive immunotherapy composition of claim 2 or 3, wherein the autoantigen-presenting cells are derived from autologous dendritic cells or B cells or a mixture thereof or peripheral blood-derived lymphocytes.

6. The adoptive immunotherapy composition of claim 2 or 3, wherein the tumor-specific T-cell receptor (TCR) is HLA-compatible or patient-specific.

7. The adoptive immunotherapy composition of claim 1, wherein autologous patient-specific T cells containing a natural T cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active population of patient-specific autologous anti-tumor T cells, said active population of patient-specific autologous anti-tumor T cells capable of promoting in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

8. The adoptive immunotherapy composition of claim 2 or 3, wherein the autologous patient-specific T cells containing a patient-specific, tumor-specific T cell receptor (TCR) are transduced with a lentiviral vector to express a chimeric antigen receptor (CAR) during or after co-culturing with autologous antigen-presenting cells transduced with one or more lentiviral vectors expressing patient-derived tumor antigens to generate an active population of patient-specific autologous anti-tumor T cells, the active population of patient-specific autologous anti-tumor T cells being able to recognize the tumor-specific T cell receptor (TCR) and being able to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, leading to tumor stabilization, reduction and / or elimination, and / or remission and / or elimination of cancer.

9. The adoptive immunotherapy composition of claim 1 or 2, wherein the patient-derived tumor antigen is identified by patient biopsy and nucleotide sequencing to identify mutant RNA transcripts within the mutant group.

10. The adoptive immunotherapy composition of claim 1 or 2, wherein the autologous anti-tumor T cell population comprises autologous antigen-presenting cells (APCs), wherein the autologous antigen-presenting cells comprise patient-specific dendritic cells or B cells or a mixture thereof or peripheral blood-derived lymphocytes.

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