CAR containing CD28 zeta and CD3 zeta

Abstract

Compositions and methods for the treatment of cancer are disclosed. The disclosure provides chimeric antigen receptors and cells expressing such chimeric antigen receptors. In certain embodiments, the engineered cells expressing the chimeric antigen receptor are specific for low-density cancer antigens or intra-groove peptide antigens.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 020177, filed May 5, 2020, which is incorporated by reference in its entirety. [Background technology]

[0002] Cancer is the second leading cause of death in the United States. Current treatments for many cancers are ineffective for certain patient populations or cause toxic side effects that significantly impact the patient's quality of life. Adoptive immunotherapy, which involves the transfer of ex vivo generated antigen-specific T cells, is a promising strategy for cancer treatment. T cells used for adoptive immunotherapy can be generated, for example, by genetically modifying T cells (e.g., by engineering them to express chimeric antigen receptors or "CARs"). However, current adoptive immunotherapy approaches are often ineffective at targeting antigens that are expressed at low density, such as peptide antigens presented by MHC class I proteins (referred to as "peptide in groove" or "PIG" antigens). Targeting PIG antigens using adoptive immunotherapy is particularly attractive for a number of reasons, including enabling the use of CAR polypeptide-expressing T cells (CAR T cells) to target intracellular cancer-associated antigens. Thus, there is a need in the art for improved adoptive immunotherapy approaches that facilitate targeting of low density antigens, including PIG antigens, for the treatment of cancer. Summary of the Invention

[0003] This application is based, at least in part, on the discovery that CAR T cells expressing a CAR comprising a CD28 / z signaling domain exhibit superior in vitro and in vivo efficacy when targeting peptide in groove (PIG) ​​antigens or low density cancer antigens. In some aspects, provided herein are CAR polypeptides and nucleic acids encoding such CAR polypeptides that comprise a CD28 / z signaling domain and target PIG antigens or low density cancer antigens, as well as methods of using and making such CAR polypeptides and nucleic acids.

[0004] In some aspects, provided herein is a CAR polypeptide comprising at least one cytoplasmic / co-stimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain. In some embodiments, the CAR polypeptide further comprises at least one cytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the CAR polypeptide further comprises an antigen binding domain specific for a low density cancer antigen and / or a groove peptide antigen.

[0005] In some embodiments, the cytoplasmic / costimulatory region of the CAR polypeptide further comprises a 4-1BB domain (e.g., in addition to the cluster of differentiation 28 zeta (CD28 / ζ) domain). The CAR polypeptide may comprise a hinge / spacer region comprising at least one cluster of differentiation 28 zeta (CD28 / ζ) domain. The CAR polypeptide may comprise a transmembrane region comprising at least one cluster of differentiation 28 zeta (CD28 / ζ) domain.

[0006] In some embodiments, a CAR polypeptide disclosed herein has antigen specificity for a cancer antigen, wherein the CAR comprises at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1-3. The CD28 / ζ sequence can be within the hinge domain, transmembrane domain, or signaling domain of the CAR. Also provided herein is a CAR polypeptide with antigen specificity for a cancer antigen, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NOs: 6-13, 29, 30, 33, or 34.

[0007] A CAR polypeptide may comprise at least one cluster of differentiation 28 (CD28) amino acid sequence that is part of the cytoplasmic / costimulatory region of the CAR. A CAR polypeptide may have at least one cluster of differentiation 28 (CD28) amino acid sequence that is part of the transmembrane region of the CAR. A CAR polypeptide may comprise at least one cluster of differentiation 28 (CD28) amino acid sequence that is part of the hinge / spacer region of the CAR.

[0008] As disclosed herein, the cancer antigen can be a groove peptide antigen and / or a low density cancer antigen. Fewer than 2,000 copies, fewer than 1,000 copies, or fewer than 500 copies of a cancer antigen may generally be found in a tumor (e.g., a solid tumor). The cancer antigen can be MAGEA4, tyrosinase, HPV16 E7, or NY-ESO peptide. The cancer antigen can be MAGEA4. 230-239 , MAGEA4 286-294 , Tyr 369-377 , HPV16E7 11-19 , or NY-ESO-1 157-165 The epitope may include at least one epitope selected from:

[0009] The CAR polypeptide can comprise a variable light chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 16-19, 31, or 37. The CAR polypeptide can comprise a variable heavy chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 20-23, 32, or 38.

[0010] In some embodiments, the present specification provides a nucleic acid that encodes a CAR polypeptide disclosed herein. The nucleic acid can be an expression vector (e.g., a viral vector, such as a lentiviral vector). The nucleic acid that encodes a CAR polypeptide can have a sequence as set forth in SEQ ID NO: 14, 15, 35, or 36.

[0011] Also provided herein are immune cells comprising a CAR polypeptide disclosed herein or nucleic acids encoding a CAR polypeptide disclosed herein. Also provided herein are compositions comprising immune cells disclosed herein and cell banks comprising such immune cells. The cell bank may be a cell bank for adoptive immunotherapy. The immune cells may be leukocytes, lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, or eosinophils. In some embodiments, the immune cells are lymphocytes selected from αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof.

[0012] In some aspects, provided herein are methods of treating cancer (e.g., a solid tumor) in a subject by administering to the subject a composition comprising a cell expressing a CAR polypeptide disclosed herein (e.g., a CAR polypeptide comprising at least one cytoplasmic / co-stimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain). In some embodiments, the CAR polypeptide further comprises at least one cytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the CAR polypeptide further comprises an antigen binding domain specific for a low density cancer antigen and / or a groove peptide antigen.

[0013] In some embodiments, the methods described herein further comprise co-administering to the subject a second CAR polypeptide comprising a 4-1BB domain in a costimulatory region of the CAR polypeptide. In some embodiments, the second CAR comprises at least one intracytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the second CAR comprises an extracellular domain specific for a low density cancer antigen and / or an intragroove peptide cancer antigen. The second CAR polypeptide may comprise a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region.

[0014] In some embodiments, the subject may have cancer. The cancer may be melanoma. [Brief description of the drawings]

[0015] [Figure 1] There are two parts, A and B, which show the general CAR construct design and the exemplary CAR constructs used in Examples 1 and 2. General BB / z and 28 / z CAR construct schematics are shown in Part A. Part B shows chimeric antigen receptors that contain, in addition to the anti-HLA-A2 / MAGEA4286-294 31345 scFv, either (top) a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain (BB / z CAR), or (middle) a huCD28 hinge / transmembrane / co-stimulatory domain, and a CD3z signaling domain (28 / z CAR). (bottom) An irrelevant scFv plus a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain served as a control CAR. [Figure 2A]There are two parts, A and B, and they show that MAGEA4286-294 28 / z CAR T cells from two donors exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4286-294 BB / z CAR T cells in the SK-MEL-37 melanoma tumor model. NSG mice were subcutaneously implanted with HLA-A2+MAGEA4+SK-MEL-37 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4286-294 BB / z CAR or anti-HLA-A2 / MAGEA4286-294 28 / z CAR from two different donors. Tumor growth was assessed by measuring tumor volume. Part A shows data for donor 1, and part B shows data for donor 2. Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 2B] There are two parts, A and B, and they show that MAGEA4286-294 28 / z CAR T cells from two donors exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4286-294 BB / z CAR T cells in the SK-MEL-37 melanoma tumor model. NSG mice were subcutaneously implanted with HLA-A2+MAGEA4+SK-MEL-37 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4286-294 BB / z CAR or anti-HLA-A2 / MAGEA4286-294 28 / z CAR from two different donors. Tumor growth was assessed by measuring tumor volume. Part A shows data for donor 1, and part B shows data for donor 2. Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 3A]There are two parts, A and B, and they show that MAGEA4286-294 28 / z CAR T cells from two donors exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4286-294 BB / z CAR T cells in the A375 melanoma tumor model. NSG mice were subcutaneously injected with HLA-A2+MAGEA4+A375 human melanoma tumor cells. 13 days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4286-294 BB / z CAR or anti-HLA-A2 / MAGEA4286-294 28 / z CAR from two different donors. Tumor growth was assessed up to day 64 by measuring tumor volume. Part A shows data for donor 1, and part B shows data for donor 2. Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 3B] There are two parts, A and B, and they show that MAGEA4286-294 28 / z CAR T cells from two donors exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4286-294 BB / z CAR T cells in the A375 melanoma tumor model. NSG mice were subcutaneously injected with HLA-A2+MAGEA4+A375 human melanoma tumor cells. 13 days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4286-294 BB / z CAR or anti-HLA-A2 / MAGEA4286-294 28 / z CAR from two different donors. Tumor growth was assessed up to day 64 by measuring tumor volume. Part A shows data for donor 1, and part B shows data for donor 2. Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 4A]There are two parts, A and B, showing that the exemplary CAR construct used in Example 3 and MAGEA4230-239 28 / z CAR T cells exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4230-239 BB / z CAR T cells in the A375 melanoma tumor model. Part A shows data from NSG mice subcutaneously injected with HLA-A2+MAGEA4+A375 human melanoma tumor cells. 13 days after tumor establishment, mice (n=4 or n=5 per group) were intravenously injected with 4x106 T cells expressing either a non-binding control BB / z CAR (control CAR T), an anti-HLA-A2 / MAGEA4230-239 BB / z CAR or an anti-HLA-A2 / MAGEA4230-239 28 / z CAR. Tumor growth was assessed up to day 37 by measuring tumor volume. The top graph shows tumor growth from days 0 to 21. The bottom graph shows tumor growth from days 0 to 37. Values ​​on the left graph represent the mean ± SEM tumor volume on the indicated days after tumor implantation. The right graph shows tumor growth curves for individual mice. Part B shows chimeric antigen receptors that contain anti-HLA-A2 / MAGEA4230-239 33229P scFv plus either (top) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR), or (middle) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR). (bottom) An irrelevant scFv plus the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as a control CAR. [Figure 4B]There are two parts, A and B, showing that the exemplary CAR construct used in Example 3 and MAGEA4230-239 28 / z CAR T cells exhibit superior in vivo antitumor activity and antitumor kinetics compared to MAGEA4230-239 BB / z CAR T cells in the A375 melanoma tumor model. Part A shows data from NSG mice subcutaneously injected with HLA-A2+MAGEA4+A375 human melanoma tumor cells. 13 days after tumor establishment, mice (n=4 or n=5 per group) were intravenously injected with 4x106 T cells expressing either a non-binding control BB / z CAR (control CAR T), an anti-HLA-A2 / MAGEA4230-239 BB / z CAR or an anti-HLA-A2 / MAGEA4230-239 28 / z CAR. Tumor growth was assessed up to day 37 by measuring tumor volume. The top graph shows tumor growth from days 0 to 21. The bottom graph shows tumor growth from days 0 to 37. Values ​​on the left graph represent the mean ± SEM tumor volume on the indicated days after tumor implantation. The right graph shows tumor growth curves for individual mice. Part B shows chimeric antigen receptors that contain anti-HLA-A2 / MAGEA4230-239 33229P scFv plus either (top) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR), or (middle) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR). (bottom) An irrelevant scFv plus the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as a control CAR. [Figure 5A]There are three parts, A-C, which show xenogeneic tumor measurements in mice administered the exemplary CAR constructs and exemplary CAR-T cells used in Example 4. Results revealed that anti-tyrosinase 369-377 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to tyrosinase 369-377 BB / z CAR T cells in WM266.4 and SK-MEL-23 melanoma tumor models. Part A shows chimeric antigen receptors that include anti-HLA-A2 / tyrosinase 369-377 D11 scFv as well as either (top) huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or (middle) huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR). (Lower panel) In addition to an irrelevant scFv, the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as control CARs. Part B shows data from NSG mice subcutaneously injected with 4x106 HLA-A2+tyrosinase+WM266.4 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 2x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume. Part C shows data from NSG mice subcutaneously injected with 5x106 HLA-A2+tyrosinase+SK-MEL-23 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume.Data are shown for donor 1 (left panel) and donor 2 (right panel). Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 5B]There are three parts, A-C, which show xenogeneic tumor measurements in mice administered the exemplary CAR constructs and exemplary CAR-T cells used in Example 4. Results revealed that anti-tyrosinase 369-377 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to tyrosinase 369-377 BB / z CAR T cells in WM266.4 and SK-MEL-23 melanoma tumor models. Part A shows chimeric antigen receptors that include anti-HLA-A2 / tyrosinase 369-377 D11 scFv as well as either (top) huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or (middle) huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR). (Lower panel) In addition to an irrelevant scFv, the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as control CARs. Part B shows data from NSG mice subcutaneously injected with 4x106 HLA-A2+tyrosinase+WM266.4 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 2x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume. Part C shows data from NSG mice subcutaneously injected with 5x106 HLA-A2+tyrosinase+SK-MEL-23 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume.Data are shown for donor 1 (left panel) and donor 2 (right panel). Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 5C]There are three parts, A-C, which show xenogeneic tumor measurements in mice administered the exemplary CAR constructs and exemplary CAR-T cells used in Example 4. Results revealed that anti-tyrosinase 369-377 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to tyrosinase 369-377 BB / z CAR T cells in WM266.4 and SK-MEL-23 melanoma tumor models. Part A shows chimeric antigen receptors that include anti-HLA-A2 / tyrosinase 369-377 D11 scFv as well as either (top) huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or (middle) huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR). (Lower panel) In addition to an irrelevant scFv, the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as control CARs. Part B shows data from NSG mice subcutaneously injected with 4x106 HLA-A2+tyrosinase+WM266.4 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 2x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume. Part C shows data from NSG mice subcutaneously injected with 5x106 HLA-A2+tyrosinase+SK-MEL-23 human melanoma tumor cells. Seven days after tumor establishment, mice (n=5 per group) were intravenously injected with 4x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / tyrosinase 369-377 BB / z CAR, or an anti-HLA-A2 / tyrosinase 369-377 28 / z CAR from two different donors. Tumor growth was assessed up to day 90 by measuring tumor volume.Data are shown for donor 1 (left panel) and donor 2 (right panel). Values ​​on the top graph represent mean ± SEM tumor volumes on the indicated days after tumor implantation. The bottom graph shows tumor growth curves for individual mice. [Figure 6A] There are two parts, A and B, which show that the exemplary CAR construct used in Example 5 and NY-ESO-1157-165 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to NY-ESO-1157-165 BB / z CAR T cells in the A375 melanoma tumor model. Part A shows a chimeric antigen receptor that includes, in addition to the anti-HLA-A2 / NY-ESO-1157-165 28105P scFv, either (top) a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain (BB / z CAR), or (middle) a huCD28 hinge / transmembrane / costimulatory domain, and a CD3z signaling domain (28 / z CAR). (Lower panel) In addition to an irrelevant scFv, the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as a control CAR. Part B shows that 5x106 HLA-A2+NY-ESO-1+A375 human melanoma tumor cells were subcutaneously injected into NSG mice. Three days after tumor establishment, mice (n=5 per group) were intravenously injected with 20x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / NY-ESO-1157-165 BB / z CAR, or an anti-HLA-A2 / NY-ESO-1157-165 28 / z CAR. Tumor growth was assessed up to day 38 by measuring tumor volume. The top graph shows tumor growth from days 0 to 21. The bottom graph shows tumor growth from days 0 to 38. Values ​​on the left graph represent the mean ± SEM tumor volume on the indicated days after tumor implantation, and graphs on the right show tumor growth curves for individual mice. [Figure 6B]There are two parts, A and B, which show that the exemplary CAR construct used in Example 5 and NY-ESO-1157-165 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to NY-ESO-1157-165 BB / z CAR T cells in the A375 melanoma tumor model. Part A shows a chimeric antigen receptor that includes, in addition to the anti-HLA-A2 / NY-ESO-1157-165 28105P scFv, either (top) a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain (BB / z CAR), or (middle) a huCD28 hinge / transmembrane / costimulatory domain, and a CD3z signaling domain (28 / z CAR). (Lower panel) In addition to an irrelevant scFv, the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain served as a control CAR. Part B shows that 5x106 HLA-A2+NY-ESO-1+A375 human melanoma tumor cells were subcutaneously injected into NSG mice. Three days after tumor establishment, mice (n=5 per group) were intravenously injected with 20x106 T cells expressing either a nonbinding control BB / z CAR (control CAR T), an anti-HLA-A2 / NY-ESO-1157-165 BB / z CAR, or an anti-HLA-A2 / NY-ESO-1157-165 28 / z CAR. Tumor growth was assessed up to day 38 by measuring tumor volume. The top graph shows tumor growth from days 0 to 21. The bottom graph shows tumor growth from days 0 to 38. Values ​​on the left graph represent the mean ± SEM tumor volume on the indicated days after tumor implantation, and graphs on the right show tumor growth curves for individual mice. [Figure 7]We show that MAGEA4286-294 28 / z CAR T cells exhibit superior in vitro cytotoxicity against various target cells compared to MAGEA4286-294 BB / z CAR T cells. In contrast to some cytotoxicity of MAGEA4+HLA-A2+A375 and SK-MEL-37 cells observed with anti-MAGEA4286-294 CAR(31345) containing the BB / z intracellular signaling domain, enhanced cytotoxicity was observed with anti-MAGEA4286-294 CAR(31345) containing the 28 / z intracellular signaling domain. Some cytotoxic effects of MAGEA4+HLA-A2+A375 and SK-MEL-37 cells were seen with anti-MAGEA4230-239 CAR (33229) containing the BB / z intracellular signaling domain, in contrast to enhanced cytotoxic effects of anti-MAGEA4239-239 CAR (33229) containing the 28 / z intracellular signaling domain, and minimal background cytotoxic effects were induced by the CTL CAR constructs. Some cytotoxic effects of MAGEA4+HLA-A2+A375 cells were seen with anti-MAGEA4230-239 CAR (34852) containing the BB / z intracellular signaling domain, in contrast to enhanced cytotoxic effects of anti-MAGEA4239-239 CAR (34852) containing the 28 / z intracellular signaling domain. [Figure 8] FIG. 1 shows a diagram of an exemplary CAR construct used in Example 6. [Figure 9]

[0023] Figure 1 shows an experimental in vivo dose-response study comparing the ability of MAGEA4(230-239) conjugate 33229 to eliminate A375 tumors when formulated as BB / z or 28 / z CARs. As shown in previous non-dose-response studies, 28 / z CAR T exhibited superior in vivo antitumor activity and antitumor kinetics. [Figure 10] FIG. 1 shows a diagram of an exemplary CAR construct used in Example 7. [Figure 11]We show that anti-HLA-A2 / MAGEA4230-239 28 / z CAR T cells exhibit superior in vivo anti-tumor activity and anti-tumor kinetics compared to anti-HLA-A2 / MAGEA4230-239 BB / z CAR T cells. Experimental parameters are discussed in Example 8. [Figure 12] FIG. 1 shows a diagram of an exemplary CAR construct used in Examples 6 and 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] overview In some aspects, provided herein are CAR polypeptides and polynucleotides encoding said CAR polypeptides, comprising at least one cytoplasmic / costimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain. The CD28 / ζ domain can be a human CD28 / ζ domain. In some embodiments, the CAR polypeptide further comprises at least one cytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. The CAR polypeptide can also comprise an antigen binding domain specific for a low density cancer antigen and / or a groove peptide antigen. Also provided herein are immune cells expressing the CAR polypeptides disclosed herein, as well as cell banks comprising immune cells expressing the CAR polypeptides disclosed herein.

[0017] As disclosed herein, cancers characterized by PIG antigens or low density cancer antigens often escape conventional cancer treatments due to their low target copy number within the tumor. In addition, solid tumors characterized by PIG or low density cancer antigens may be more resistant to CAR-T therapy and more difficult to treat due to the presence of cancer-associated peptides in the grooves within the tumor, rather than cell surface antigens. Thus, there is a need for improved CAR therapies provided herein that can effectively target PIG and / or low density cancer antigens to improve / enhance the efficacy of CAR therapy for cancers, particularly cancers characterized by solid tumors.

[0018] The methods disclosed herein include methods of treating a tumor in a subject by administering a composition comprising cells expressing a CAR polypeptide disclosed herein. In some embodiments, the CAR polypeptide disclosed herein has specificity for low density cancer antigens or PIG antigens. Low density cancer antigens include, but are not limited to, any cancer antigen in which less than 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 copies of the cancer antigen are typically found in solid tumors. The cancer antigen can be MAGEA4, tyrosinase, HPV16 E7, or NY-ESO peptide. The cancer antigen can be MAGEA4, tyrosinase, HPV16 E7, or NY-ESO peptide. 230-239 , MAGEA4 286-294 , Tyr 369-377 , HPV16E7 11-19 , or NY-ESO-1 157-165 For example, the CAR may comprise an antigen binding domain using the variable light and / or heavy chains of an antibody specific for a low density or PIG antigen, such as an epitope sequence of MAGEA4, tyrosinase, HPV16 E7, or NY-ESO peptide, or a portion thereof.

[0019] definition For convenience, certain terms employed in the specification, examples, and appended claims are listed here.

[0020] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] As used herein, the term "administration" means providing a pharmaceutical agent or composition to a subject, including, but not limited to, administration by a medical professional and self-administration. Such agents can include, for example, CAR T cells provided herein.

[0022] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acidic functional groups and that are capable of being included in a natural amino acid polymer. Exemplary amino acids include the natural amino acids; their analogs, derivatives and congeners; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0023] The term "binding" or "interaction" refers to a stable association between two molecules, e.g., a peptide and a binding partner or agent, e.g., a small molecule, which may result, for example, from electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions.

[0024] As used herein, the term "cancer" includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels, including those of the cervix, anus, vagina, vulva, penis, base of tongue, larynx, and tonsils. The term "cancer" further encompasses primary and metastatic cancers.

[0025] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, e.g., an antibody-based specificity for an antigen of interest (e.g., a tumor antigen), with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cell immune activity. In general, CARs consist of an extracellular single-chain antigen binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0026] "Costimulatory domain" or "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. The costimulatory domain can be a human costimulatory domain. Exemplary costimulatory molecules include CD28, 4-1BB, CD27, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0027] "Costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation activation, differentiation, etc. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3.

[0028] A "costimulatory signal" refers to a signal that, in combination with a primary signal, leads to T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0029] The term "epitope" refers to a protein determinant that can specifically bind to an antibody or immune cell (e.g., a T cell). Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains. A particular epitope can be defined by a particular sequence of amino acids to which a CAR or antibody can bind.

[0030] "Genetic construct" refers to a nucleic acid, such as a vector, plasmid, viral genome, etc., that contains a "coding sequence" for a polypeptide or that is otherwise capable of being transcribed into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), transfected into a cell, e.g., a mammalian cell, and causing expression of the coding sequence in a cell transfected with the construct. A genetic construct can include one or more regulatory elements operably linked to the coding sequence, as well as intervening sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0031] The terms "ligand binding domain" and "antigen binding domain" are used interchangeably herein and refer to the portion of a chimeric antigen receptor that specifically binds to a given antigen. The term "linker" is art-recognized and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. A linker may consist of a single linking molecule or may include a linking molecule and a spacer molecule whose purpose is to separate the linking molecule and the compounds by a specific distance.

[0032] The term "operably linked" refers to a functional relationship between a nucleic acid and another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operably linking a DNA to a transcriptional regulatory element refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0033] As used herein, the phrase "pharmacologically acceptable" refers to agents, compounds, materials, compositions and / or formulations that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of safe medical judgment and commensurate with a reasonable benefit / risk ratio.

[0034] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as glyceryl stearate, glyceryl stearate, and glyceryl stearate; and (11) glyceryl stearate. Examples of suitable non-toxic and compatible materials include: (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic and compatible materials used in pharmaceutical formulations.

[0035] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to natural or synthetic molecules, or some combination thereof, that contain a single nucleotide, or two or more nucleotides linked at the 3' position of one nucleotide to the 5' end of another nucleotide by a phosphate group. Polymeric forms of nucleotides are not limited in length and may contain deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and perform any function. Non-limiting examples of polynucleotides are: coding or non-coding regions of a gene or gene fragment, locus(es) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides can be replaced with T nucleotides. A polynucleotide is not necessarily associated with the cell in which the nucleic acid occurs in nature and / or is not operably linked to the polynucleotide it is naturally linked to.

[0036] The term "precancerous lesion" or "precancerous pathology" refers to atypical cells and / or tissues that are associated with an increased risk of cancer. The term "precancerous lesion" can refer, for example, to dysplasia, benign tumor formation, or carcinoma in situ.

[0037] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0038] The "signal transduction domain" or "signal transduction domain" of a CAR as used herein is responsible for intracellular signal transduction after the extracellular ligand binding domain binds to a target, thereby resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signal transduction domain" refers to a portion of a protein that transduces an effector function signal and induces the cell to perform its intrinsic function. Examples of signal transduction domains for use in CARs may be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after antigen receptor ligation, as well as any derivative or variant of these sequences, and any synthetic sequence with the same functional capabilities. In some cases, the signal transduction domain includes two different classes of cytoplasmic signal transduction sequences, those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences may include signaling motifs known as ITAMs, immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0039] "Spacer" as used herein refers to a peptide that connects proteins (e.g., in a fusion protein). In general, spacers have no special biological activity other than connecting proteins together or maintaining some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer can be selected to affect some properties of the molecule, such as the folding, net charge, or hydrophobicity of the molecule.

[0040] The term "specifically binds" or "specific binding" as used herein, when referring to a polypeptide (including a CAR polypeptide), refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous mixture of proteins and other biological products. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen) when it does not bind in significant amounts to other proteins present in the sample or to other proteins that the ligand or antibody may come into contact with in the organism. In general, a first molecule that "specifically binds" to a second molecule will bind to the second molecule in a manner that is consistent with the above-mentioned binding reactions, and may be useful in determining whether the first molecule binds to the second molecule or not. 5 M -1 More than (e.g., 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1For example, in terms of the ability of a PIG-specific CAR to bind a peptide presented on an MHC (e.g., class I MHC or class II MHC), the CAR typically binds specifically to that peptide / MHC with an affinity of at least about 10-4 M or less, KD, and binds to a given antigen / binding partner with an affinity (expressed in KD) that is at least 10-fold, at least 100-fold, or at least 1000-fold lower than the affinity with which it binds to a non-specific and unrelated peptide / MHC complex (e.g., one that includes a BSA peptide or a casein peptide).

[0041] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy. The terms "transformation," "transfection," or "transduction" refer to the introduction of a nucleic acid, such as an expression vector, into a recipient cell (eg, a mammalian cell) and includes the introduction of the nucleic acid into the chromosomal DNA of said cell.

[0042] As used herein, the term "treatment" refers to a clinical intervention designed to modify the natural course of the individual being treated during the course of clinical disease.The desired effects of treatment include slowing down, alleviating or reducing the rate of progression of pathological conditions, as well as remission or improvement of prognosis of a particular disease, disorder or condition.For example, an individual is considered to be "treated" successfully if one or more symptoms associated with a particular disease, disorder or condition are alleviated or eliminated.

[0043] The term "variant" refers to amino acid or peptide sequences having conservative amino acid substitutions, non-conservative amino acid substitutions (e.g., degenerate variants), substitutions within the wobble positions of each codon encoding an amino acid (e.g., in DNA and RNA), amino acids added to the C-terminus of a peptide, or peptides with 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0044] The term "vector" refers to a means by which a nucleic acid may be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, to which a nucleic acid may be linked and which may or may not be capable of autonomous replication or which may or may not be capable of integrating into a host cell chromosome. Such vectors may include any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0045] In certain embodiments, an agent may be used alone or may be co-administered with another type of therapeutic agent. As used herein, the phrase "co-administration" or "co-administered" refers to any form of administration of two or more different therapeutic agents, such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., such that the two agents are effective simultaneously in a subject, which may include a synergistic effect of the two agents). For example, the different therapeutic agents may be administered simultaneously or sequentially, in the same formulation or in separate formulations. In certain embodiments, the different therapeutic agents may be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week of each other. Thus, subjects receiving such treatment may benefit from the combined effects of the different therapeutic agents.

[0046] Chimeric antigen receptors (CARs) It has been previously successful to generate novel specificities in T cells through genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are receptors that contain a targeting moiety that binds to one or more signaling and / or costimulatory domains in a single fusion molecule. In certain embodiments, the binding moiety of the CAR comprises the antigen-binding domain of a single-chain antibody (scFv), which comprises the light and heavy chain variable fragments of a monoclonal antibody linked by a flexible linker. In certain embodiments, the binding moiety further comprises the transmembrane and hinge domains of a monoclonal antibody.

[0047] In certain aspects, the CAR polypeptides provided herein comprise at least one cytoplasmic / costimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain. In addition, the hinge / spacer region and / or transmembrane region of the CAR, or the transmembrane region of the CAR, may comprise a CD28 / ζ domain. The CAR may comprise at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1-3. The CAR polypeptides disclosed herein may comprise at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NOs: 1-3. The CAR polypeptide may comprise all three sequences set forth in SEQ ID NOs: 1-3. For example, the CAR hinge domain may comprise SEQ ID NO:2 and the costimulatory domain may comprise SEQ ID NO:3.

[0048] [Table 1]

[0049] In some embodiments, the CAR comprises an antigen binding domain specific for a low density cancer antigen and / or an intragroove peptide antigen. Exemplary PIG antigens and low density cancer antigens can be found in Table 1. In some embodiments, the CAR polypeptide disclosed herein specifically binds to an epitope listed in Table 2. In some embodiments, the present disclosure provides a melanoma associated antigen A4 (MAGE-A4)-specific chimeric antigen receptor (CAR), wherein the MAGE-A4-specific chimeric antigen receptor interacts with amino acids 286-294 of SEQ ID NO:4, i.e., a portion thereof. The polypeptide sequence of MAGE-A4 (286-294) is shown in SEQ ID NO:5.

[0050] [Table 2]

[0051] [Table 3]

[0052] Further exemplary PIG epitopes include melanoma associated antigen 3 (MAGE-A3); melanoma associated antigen 1 (MAGE-1); melanoma associated antigen 10 (MAGEA10); melanoma antigen recognized by T cells 1 (MART-1), Epstein-Barr virus (EBV) latent membrane protein (LMP2); mouse double minute 2 (MDM2); melanoma associated antigen 1 (MAGE-A1); glycoprotein 100 (gp100); tumor protein p53 (P53); minor histocompatibility antigen (mHag); minor histocompatibility antigen HA-1 (HA-1); ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome (UTY); ribosomal protein S4, Y-linked (RPS4Y); It may be derived from MHC class II restricted DEAD-box RNA helicase Y (DBY); cytotoxic T lymphocyte (CTL)-recognized antigen of melanoma (CAMEL); Wilms' tumor 1 (WT1); renal cell carcinoma (RCC) tumor antigen; murine mastocytoma P815; leucine zipper protein 4 (LUZP4); cancer / testis-associated SPANX antigen; ATPase family AAA domain-containing protein 2 (ATAD2); Rhox homeobox family member 2 (RHOXF2); cancer / testis antigen 136; F-Box protein 39 (FBXO39); TDRD4; WW domain-binding protein 2 N-terminal-like (WBP2NL) or carcinoembryonic antigen (CEA).

[0053] Also provided herein is a CAR polypeptide comprising an amino acid sequence set forth in SEQ ID NO:6-13, 29, 30, 33, or 34. The CAR polypeptide disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NO:6-13, 29, 30, 33, or 34.

[0054] [Table 4-1]

[0055] [Table 4-2]

[0056] [Table 4-3]

[0057] [Table 4-4]

[0058] [Table 4-5]

[0059] [Table 4-6]

[0060] [Table 4-7]

[0061] [Table 4-8]

[0062]

Table 4-9

[0063]

Table 4-10

[0064]

Table 4-11

[0065]

Table 4-12

[0066]

Table 5-1

[0067]

Table 5-2

[0068]

Table 5-3

[0069]

Table 5-4

[0070]

Table 5-5

[0071]

Table 5-6

[0072] [Table 5-7]

[0073] [Table 5-8]

[0074] [Table 5-9]

[0075] [Table 5-10]

[0076] In certain embodiments, the binding domain and / or extracellular domain of the CARs provided herein provide a CAR with the ability to bind to a target antigen of interest. A binding domain (e.g., a ligand-binding domain or an antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and specifically bind to a biological molecule (e.g., a cell surface receptor or a tumor protein, or a component thereof). A binding domain includes any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, as further described herein, a binding domain can be an antibody light and heavy chain variable region, and the light and heavy chain variable regions can be single chains, in either orientation (e.g., V L -V H or V H -V L) may be linked together. Various assays are known to identify binding domains of the present disclosure that specifically bind to a particular target, such as Western blot, ELISA, flow cytometry, or surface plasmon resonance (e.g., using BIACORE analysis). The target may be a clinically significant antigen where it is desirable to induce an effector immune response that results in tumor killing. The CAR polypeptide may comprise a variable light chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 16-19, 31, or 37. The variable light chain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NOs: 16-19, 31, or 37. The CAR polypeptide may comprise a variable heavy chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 20-23, 32, or 38. The variable heavy chain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NO:20-23, 32 or 38.

[0077] [Table 6]

[0078] [Table 7]

[0079] [Table 8-1]

[0080] [Table 8-2]

[0081] In one embodiment, the binding domain of the CAR is a single chain antibody (scFv) specific for a groove peptide or a low density cancer antigen, and may be a murine, human, or humanized scFv. The single chain antibody may be cloned from a V region gene of a hybridoma specific for a desired target. Techniques that can be used for cloning the variable heavy chain (VH) and variable light chain (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but may also be a non-antibody-derived binding domain. The antibody-derived binding domain may be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, where the fragment is responsible for binding to the antigen.

[0082] In certain embodiments, the CAR of the present disclosure may include linkers between the various domains, added for proper spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the binding domains VH or VL, which may be 1-10 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1-20 or 20 amino acids in length. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Linkers of ranges inclusive of the numbers described herein, for example, 10-30 amino acids in length, are also included herein.

[0083] In certain embodiments, a linker suitable for the CAR described herein is a flexible linker. Suitable linkers can be readily selected and can be of any different suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0084] Exemplary flexible linkers include glycine polymers (G)n (n is at least an integer), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has significantly more access to the Φ-Ψ space than alanine and is much less restrictive than longer side chain residues. Those skilled in the art will appreciate that CAR designs may include fully or partially flexible linkers, such that the linker can include one or more moieties that confer a less flexible structure, as well as flexible linkers to provide the desired CAR structure.

[0085] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that distances the antigen binding domain from the effector cell surface, allowing for proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6:412-419). The hinge region of a CAR is usually between the transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or modified.

[0086] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. In some embodiments, the transmembrane domain may be a CD3ζ transmembrane domain. Other transmembrane domains that can be used in some embodiments include those derived from CD8, CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine.

[0087] In certain embodiments, the CARs provided herein comprise an intracellular signaling domain. The intracellular signaling domain (also referred to herein as a "signaling domain") comprises a portion of a chimeric antigen receptor protein that is involved in transmitting the message of effective CAR binding to a target antigen to the interior of an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0088] In certain embodiments, the CARs provided herein comprise one or more immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs that contain useful primary cytoplasmic signaling sequences include those derived from TCRzeta, FcR gamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain of the CARs described herein is derived from CD3zeta.

[0089] In certain embodiments, the CARs provided herein further comprise a costimulatory domain. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a secondary signal necessary for efficient activation and function of T lymphocytes upon antigen binding. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands that specifically bind to CD83. Thus, the present disclosure further provides an exemplary costimulatory domain derived from CD28. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells expressing the CAR receptor. Also disclosed herein are CAR polypeptides in which the cytoplasmic / costimulatory region of the CAR polypeptide further comprises a 4-1BB domain (e.g., in addition to the CD28 / zeta domain). The costimulatory region of such a CAR polypeptide can comprise the complete 4-1BB domain or a fragment thereof, and / or the complete CD28 / ζ domain or a fragment thereof. The intracellular signaling and costimulatory signaling domains can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0090] The CAR polypeptide sequences disclosed herein may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 26-28. The CAR polypeptides disclosed herein may comprise at least an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NOs: 26-28.

[0091] [Table 9]

[0092] Nucleic Acids and Vectors In certain aspects, nucleic acid and polynucleotide vectors encoding the CAR polypeptides disclosed herein are also disclosed.

[0093] The nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art, such as by screening libraries of cells expressing the gene, by deriving the gene from a vector known to contain it, or by direct isolation from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be made synthetically rather than cloned.

[0094] Expression of a nucleic acid encoding a CAR is usually achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0095] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. A vector is a vehicle into which a polynucleotide encoding a protein can be covalently inserted to cause expression of the protein and / or cloning of the polynucleotide. Such vectors are sometimes referred to as "expression vectors". An isolated polynucleotide can be inserted into a vector using any suitable method known in the art, for example, but not limited to, a vector can be digested with an appropriate restriction enzyme and then ligated with the isolated polynucleotide having compatible restriction ends. An expression vector has the ability to incorporate and express heterologous or modified nucleic acid sequences that code for at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. An expression vector may contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well, which are discussed below. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, allowing it to be maintained in two organisms, for example, in a human cell for expression and in a prokaryotic host for cloning and amplification.

[0096] The expression vector can have the necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences, such as CMV, PGK and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and 3'UTR AAUAAA transcription termination sequences for efficient gene transcription and translation in its corresponding host cell. Other suitable promoters include the constitutive promoter of the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, may also be used. In certain embodiments, inducible promoters are also contemplated as part of the vector expressing the chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0097] Expression vectors can have additional sequences such as 6x-histidine, c-Myc, and FLAG tags that are incorporated into the expressed CAR. Thus, expression vectors can be engineered to include 5' and 3' untranslated regulatory sequences that sometimes function as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest contained in the expression vector. Expression vectors can also be engineered for replication and / or expression functions (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors may also include selection markers to maintain the vector in a host or recipient cell.

[0098] In various embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), bacteriophages (such as lambda phage or M13 phage), and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequence of the CAR disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0099] In certain embodiments, the nucleic acid encoding the CAR is provided in a viral vector. The viral vector may be derived, for example, from a retrovirus (e.g., foamy virus) or lentivirus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector may contain coding sequences for various chimeric proteins described herein in place of non-essential viral genes. The vectors and / or particles may be utilized to transcribe DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0100] In certain embodiments, the viral vector comprising the coding sequence of the CAR described herein is a retroviral vector or a lentiviral vector.The term "retroviral vector" refers to a vector that comprises structural and functional genetic elements that are primarily derived from retroviruses.The term "lentiviral vector" refers to a vector that comprises structural and functional genetic elements outside the LTR that are primarily derived from lentiviruses.

[0101] Retroviral vectors as used herein may be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retrovirus" also includes the lentivirus family of retroviruses, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, as well as human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0102] As used herein, lentiviral vector refers to a vector derived from lentivirus, a group (or genus) of retroviruses that cause slowly progressing diseases. Viruses in this group include HIV (human immunodeficiency virus; HIV type 1 and HIV type 2); Visna-Maedi; Caprine arthritis-encephalitis virus; Equine infectious anemia virus; Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). Preparation of recombinant lentivirus can be achieved using the method by Dull et al. and Zufferey et al. (Dull et al., J.Virol., 1998; 72:8463-8471 and Zufferey et al., J.Virol. 1998; 72:9873-9880).

[0103] The retroviral vectors (i.e., both lentiviral and non-lentiviral) used can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, FM et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 85:3014-3018; 87:6141-6145;Huber et al.(1991)Proc.Natl.Acad.Sci.USA 88:8039-8043;Ferry et al.(1991)Proc.Natl.Acad.Sci.USA 88:8377-8381;Chowdhury et al.(1991)Science 254:1802-1805;van Beusechem et al.(1992)Proc.Natl.Acad.Sci.USA 89:7640-7644;Kay et al.(1992)Human Gene Therapy3:641-647;Dai et al.(1992)Proc.Natl.Acad.Sci.USA 89:10892-10895;Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application No. WO89 / 07136; PCT Application No. WO89 / 02468; PCT Application No. WO89 / 05345; and PCT Application No. WO92 / 07573).

[0104] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for vector formation include, for example, genomic RNA and cDNA available from commercial sources such as Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0105] For the expression of CAR, a vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. The expression vector may include various elements for controlling expression, such as, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection marker, and a signal sequence. These elements can be appropriately selected by those skilled in the art, as described above. For example, a promoter sequence can be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence can be selected to enhance transcription of a polynucleotide. A selection marker can be selected to allow selection of a host cell into which the vector has been inserted from a host cell that has not, for example, a selection marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow transport of the expressed polypeptide out of the host cell.

[0106] For cloning a polynucleotide, a vector may be introduced into a host cell (isolated host cell) to allow the vector to replicate itself and thereby amplify copies of the polynucleotide contained in the vector. A cloning vector may generally contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements can be appropriately selected by those skilled in the art. For example, an origin of replication can be selected to promote autonomous replication of the vector in a host cell.

[0107] In certain embodiments, the present disclosure provides an isolated host cell comprising the vector provided herein. The host cell comprising the vector may be useful for expressing or cloning the polynucleotide contained within the vector. Suitable host cells may include, but are not limited to, higher eukaryotic cells, such as prokaryotic cells, fungal cells, yeast cells, or mammalian cells. Prokaryotic cells suitable for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacter, such as Escherichia, such as E. Coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as erratia marcescan, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa and Streptomyces.

[0108] The CAR is introduced into the host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid may be maintained transiently or stably introduced. Transfection can be performed by a variety of methods known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistec. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by viral infection instead of transfection. In certain embodiments, a retroviral vector is transduced by packaging the vector into virions prior to contact with the cell. For example, a nucleic acid encoding a CAR carried by a retroviral vector can transduce cells via infection and proviral integration.

[0109] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells can include either a selection marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells transfected or infected via a viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be located next to the appropriate regulatory sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes.

[0110] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present or expressed in the recipient organism or tissue and encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after the DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, constructs containing the minimal 5' flanking region that exhibits high levels of expression of the reporter gene are identified as promoters. Such promoter regions may be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-induced transcription.

[0111] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0112] A representative delivery vehicle in cases where a non-viral delivery system is utilized is a liposome. In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that is bound to both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles or in bilayer structures with a "collapsed" structure. They may also simply be interspersed in the solution, forming aggregates that are sometimes not uniform in size or shape. The lipid is a lipid substance that may be a natural lipid or a synthetic lipid. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as classes of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, Missouri; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, New York); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Alabama).

[0113] Immune effector cells In certain aspects, immune effector cells engineered to express the disclosed CAR polypeptides are also disclosed herein. In some embodiments, the cells can be harvested from the subject to be treated (i.e., autologous). However, in certain embodiments, immune effector cell lines or donor effector cells (allogeneic) are used.

[0114] Immune effector cells can be harvested from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural fluid, spleen tissue, and tumors. Immune effector cells can be harvested from blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be harvested by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing mononuclear cells, for example, by centrifugation through a PERCOLL™ gradient, or by convection centrifugal elutriation. Specific subpopulations of immune effector cells may be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies against surface markers specific to cells that have undergone positive selection, for example, by incubation with antibody-conjugated beads for a time interval sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cell populations can be achieved by negative selection using a combination of antibodies against surface markers unique to the cells that have undergone negative selection.

[0115] The present disclosure provides a method for generating immune effector cells expressing a CAR as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having tumor cells expressing PIG and / or low density cancer antigens, such that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically engineered without further manipulation in vitro. Such cells can then be readministered directly to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically engineered to express a CAR. In this regard, the immune effector cells can be cultured before or after being genetically engineered (i.e., transduced or transfected to express a CAR as described herein).

[0116] Prior to in vitro manipulation or genetic modification of immune effector cells as described herein, a source of cells can be harvested from a subject. In particular, immune effector cells for use with the CARs described herein include T cells. T cells can be harvested from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be harvested from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood are harvested by apheresis. The apheresis product typically contains lymphocytes, including T cells, mononuclear cells, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and the cells placed in a buffer or medium appropriate for further processing. In one embodiment, the cells are washed with PBS. In alternative embodiments, the washed solution lacks calcium and may lack magnesium, or may lack many, but not all, divalent cations. As will be appreciated by those of skill in the art, the washing step may be performed by methods known to those of skill in the art, such as using a semi-automated flow centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffering agents. In certain embodiments, undesirable components of the apheresis sample may be removed with the medium in which the cells are directly resuspended.

[0117] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing mononuclear cells, e.g., by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved by a combination of antibodies against surface markers unique to the cells that have undergone negative selection. One method used herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells that have undergone negative selection. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest.

[0118] PBMCs may be used directly for genetic modification with CARs using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells can be harvested by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each type of CD8+ cell. In embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0119] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be harvested by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L and CD45RO negative.

[0120] Immune effector cells such as T cells can be genetically engineered after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically engineered. In another embodiment, immune effector cells such as T cells are genetically engineered with a chimeric antigen receptor described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Pat. No. 6,905,874; U.S. Pat. No. 6,867,041; U.S. Pat. No. 6,797,514; WO2012079000. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in a culture medium containing a suitable cytokine, such as IL-2 (e.g., recombinant human IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as "surrogate" antigen presenting cells (APCs). In other embodiments, T cells can be activated and stimulated to grow with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177; U.S. Patent No. 5,827,642; and WO2012129514.

[0121] In some embodiments, immune effector cells include any white blood cells involved in defending the body against infections and foreign substances. For example, immune effector cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells may include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0122] T helper cells (T H CD4 T cells (CD4 subunits) assist other white blood cells in immune processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore referred to as CD4 + Also known as T cells, helper T cells become activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Upon activation, they divide rapidly and secrete small proteins called cytokines that regulate or support an active immune response. These cells are called T H 1. T H 2. T H 3. T H 17, T H 9, or T FH They can differentiate into one of several subtypes, including those that secrete different cytokines and promote different types of immune responses.

[0123] Cytotoxic T cells (T C CTLs (cytotoxic T cells, or CTLs) destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore known as CD8 + Also known as T cells. These cells recognize their targets by binding antigens associated with MHC class I molecules present on the surface of all nucleated cells. They recognize the antigens of the CD8 T cells through IL-10, adenosine, and other molecules secreted by regulatory T cells. +The cells are inactivated into an anergic state, thereby preventing autoimmune disease.

[0124] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has cleared. They rapidly expand into large numbers of effector T cells upon reexposure to their cognate antigen, thereby providing the immune system with a "memory" of past infections. Memory cells are CD4 + or CD8 + Memory T cells normally express the cell surface protein CD45RO.

[0125] Regulatory T cells (T cells), formerly known as suppressor T cells, reg CD4 T cells are crucial for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. + T reg Two main classes of cells, natural T reg Cells and adaptive T reg The cells have been previously described.

[0126] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0127] In some embodiments, the T cells are CD4 + In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T composition includes cytotoxic CD8 + They are T lymphocytes.

[0128] Natural killer (NK) cells are CD56 receptor markers that can kill virus-infected and transformed cells and constitute an important cell subset of the innate immune system. + CD3 - It is a large granular lymphocyte (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). cytotoxic CD8 + Unlike T lymphocytes, NK cells can initiate cytotoxic effects against tumor cells without the need for prior sensitization and eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they avoid the potentially fatal complications of cytokine storm (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target and off-tumor effects.

[0129] Binding properties of chimeric antigen receptors As used herein, the term "binding" in the context of chimeric antigen receptor binding is to a given antigen, e.g., a cell surface protein or fragment thereof (or to an antigen that binds to a cell surface protein, such as an HLA molecule). Binding typically refers to an interaction or association between at least two independent entities or molecular structures, such as an antigen-binding domain:antigen interaction. For example, binding affinity is typically on the order of about 10, as measured by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using, for example, an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or antiligand). -8 mol / L(10 -8 M) or less, about 10 -9 mol / L(10 -9 M) or less, such as about 10 -7 mol / L(10 -7 M) K below DCell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlates strongly with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0130] Thus, in some embodiments, the chimeric antigen receptor of the present disclosure has a K that is at least 10-fold lower than the affinity with which it binds to a non-specific antigen (e.g., BSA, casein). D The chimeric antigen receptor of the present disclosure binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value. As described herein, the chimeric antigen receptor of the present disclosure is capable of binding to an HLA-presented antigen as described herein. In accordance with the present disclosure, in some embodiments, the K value is 10-fold lower than that of a non-specific antigen. D A chimeric antigen receptor affinity of this value may be considered as undetectable binding.

[0131] The term “K D " (M) refers to the dissociation equilibrium constant of a particular antigen-binding domain:antigen interaction. D Since there is a reciprocal relationship between binding affinity and K D The smaller the value, the higher, i.e., the stronger, the affinity. Thus, the term "higher affinity" or "stronger affinity" refers to a higher ability to form an interaction, hence the K D A lower affinity means a lower ability to form an interaction, and conversely, the term "lower affinity" or "weaker affinity" means a lower K D In some contexts, a higher binding affinity (or K ) of a particular molecule (e.g., a chimeric antigen receptor) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., a chimeric antigen receptor) to another interaction partner molecule (e.g., antigen Y) can refer to a higher binding affinity (or K ) of the molecule (e.g., a chimeric antigen receptor) to its interaction partner molecule (e.g., antigen Y). D ) is larger than K DA smaller K value (i.e., weaker affinity) is D The binding affinity can be expressed as a ratio determined by dividing by the value (the higher, ie, stronger affinity), for example, 5-fold or 10-fold greater binding affinity, as the case may be.

[0132] The term "k" d " (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antigen-binding domain:antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. The above values ​​are also known as k off Also called value.

[0133] The term "k" a " (M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antigen-binding domain:antigen interaction, or the association rate constant of a chimeric antigen receptor. The term “K A " (M-1 or 1 / M) refers to the association equilibrium constant of a particular antigen-binding domain:antigen interaction, or the association equilibrium constant of a chimeric antigen receptor. The association equilibrium constant is k a k d This is obtained by dividing by

[0134] The term "EC50" or "EC 50 " refers to the half-maximal effective concentration, which includes the concentration of chimeric antigen receptor that induces a response halfway between baseline and maximum after a given exposure time. EC 50 essentially represents the concentration of chimeric antigen receptor at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of a chimeric antigen receptor of the present disclosure that gives half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen) as measured, for example, by a FACS binding assay. Thus, the EC 50 As the half-maximal effective concentration value increases, decreased or weaker binding is observed.

[0135] In one embodiment, the reduction in binding is measured by measuring the chimeric antigen receptor concentration, EC50 can be defined as an increase in The present disclosure provides high affinity (e.g., nanomolar or subnanomolar K D The present invention provides a chimeric antigen receptor comprising an antigen-binding domain derived from an antibody that binds to a human antigen at a specific target site (a specific target site).

[0136] According to certain embodiments, the present disclosure provides a method for the preparation of a medicament having a K of less than about 5 nM as measured by surface plasmon resonance. D In certain embodiments, the chimeric antigen receptor comprises an antigen-binding domain derived from a corresponding antibody that binds to a human antigen at (e.g., at 25° C.) at 100° C. In certain embodiments, the corresponding antibody has a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or about 25 pM, as measured by surface plasmon resonance. D It binds to antigen proteins.

[0137] The present disclosure also provides chimeric antigen receptors comprising an antigen binding domain derived from a corresponding antibody that binds to an antigen protein with a dissociation half-life (t1 / 2) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25° C. In certain embodiments, the corresponding antibody binds to an antigen protein with a t1 / 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25° C.

[0138] The present disclosure also provides a chimeric antigen receptor comprising an antigen binding domain derived from a corresponding antibody that specifically binds to a human cell line expressing endogenous MAGE-A4 as measured by a FACS binding assay.

[0139] Treatment method The immune effector cells expressing the CAR disclosed herein induce a therapeutically beneficial immune response against cancer cells expressing PIG or low density antigens. For example, the anti-tumor immune response induced by the disclosed CAR-modified immune effector cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy approach in which the CAR-modified immune effector cells induce an immune response specific to PIG or low density cancer antigens.

[0140] CAR-expressing immune effector cells prepared as described herein can be utilized in adoptive immunotherapy methods and compositions according to known techniques, or variations thereof that will be apparent to those of skill in the art based on this disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; also U.S. Patent No. 4,690,915 to Rosenberg.

[0141] In some aspects, provided herein are methods of treating cancer (e.g., solid tumors) in a subject by administering to the subject a composition comprising cells expressing a CAR polypeptide disclosed herein. In some embodiments, the methods described herein further comprise co-administering to the subject a composition comprising cells expressing a second CAR polypeptide comprising a 4-1BB domain in a costimulatory region of the CAR polypeptide. In some embodiments, the second CAR comprises at least one intracytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the second CAR comprises an extracellular domain specific for a low density cancer antigen and / or an intragroove peptide cancer antigen. The second CAR polypeptide may comprise a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region. Without wishing to be bound by theory, immune cells expressing the first CAR (i.e., a CAR polypeptide that includes a CD28 / zeta domain in the costimulatory domain of the CAR) provide an initial burst that rapidly promotes cancer cell killing, while administration of immune cells with 4-1BB provides sustained cancer cell killing, albeit at a lower level of killing compared to administration of immune cells expressing the first CAR. The 4-1BB / CD3z CAR is associated with persistence of the patient's CAR T cells, which results in sustained cancer cell killing.

[0142] In some embodiments, the cells are formulated by first harvesting them from the culture medium, then washing the cells and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutical acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically standard saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0143] A therapeutically effective amount of cells in a composition is at least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset) or typically includes more than 102, up to 106, and 108 or 109 cells, and can be more than 1010 cells. The number of cells will vary depending on the end use for which the composition is intended, as will the types of cells included in the composition.

[0144] The cells may be autologous or xenogeneic to the patient receiving the treatment. The cells may be allogeneic. Optionally, the treatment may also include administration of mitogens (e.g., PHA), or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.), as described herein, to enhance induction of an immune response.

[0145] The immune effector cell population expressing a CAR can be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. The pharmaceutical compositions disclosed herein can include an immune effector cell population expressing a CAR, such as T cells as described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, such as neutral buffered saline, phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions disclosed herein can be formulated for intravenous administration.

[0146] The anti-tumor immune response induced in a subject by administering T cells expressing a CAR described herein using the methods described herein or other methods known in the art may include cytotoxic T cells, which can kill infected cells, cellular immune responses mediated by regulatory T cells, and helper T cell responses. A humoral immune response mediated primarily by helper T cells, which can activate B cells, thereby resulting in antibody production, may also be induced. Various techniques can be used to analyze the type of immune response induced by the compositions disclosed herein and are fully described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0147] Accordingly, provided herein is a method of treating an individual diagnosed or suspected of having or being at risk for developing a malignant tumor, the method comprising administering to the individual a therapeutically effective amount of an immune effector cell expressing a CAR as described herein.

[0148] Administration of the disclosed compositions can be performed in any convenient manner, including injection, infusion, or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (iv) or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions can also be injected directly into a tumor, lymph node, or site of infection.

[0149] Provided herein is a method of co-administering to a subject a second CAR polypeptide comprising a 4-1BB domain in a costimulatory region of the CAR polypeptide. The second CAR polypeptide may further comprise at least one intracytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain and / or an extracellular domain specific for a low density cancer antigen and / or an intragroove peptide cancer antigen. The second CAR polypeptide may comprise a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region.

[0150] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to patients in combination with (e.g., before, simultaneously, or after) any number of relevant therapies, including but not limited to additional cancer therapies. In some embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodepleting agents such as CAM PATH, anti-CD3 antibodies or other antibody therapeutics, cytoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. In some embodiments, the CAR-modified immune effector cells are administered to patients in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, T cell depleting therapy using any chemotherapy agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In other embodiments, the cell composition is administered after B cell depletion therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in some embodiments, the subject can receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells. In another embodiment, the expanded cells are administered before or after surgery to treat the subject's cancer or precancerous lesion.

[0151] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of engineered cells may be administered to a subject over a defined time course. The method according to this aspect includes sequentially administering multiple doses of cells to a subject. As used herein, "sequentially administering" means that each dose is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure provides a method that includes sequentially administering a single initial dose to a patient, followed by one or more secondary doses, and optionally one or more tertiary doses thereafter.

[0152] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the engineered cells of the present disclosure. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of engineered cells, but generally differ from one another by the frequency of administration. However, in certain embodiments, the amount of engineered cells contained in the initial, secondary, and / or tertiary doses varies from one another (e.g., adjusted up or down appropriately) during the treatment period. In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered at the beginning of a treatment regimen as a "loading dose", followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0153] In one embodiment of the disclosure, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or more) after the immediately preceding dose. The phrase "immediately preceding dose" as used herein means a dose in a multiple administration series that is administered to a patient prior to administration of the next dose in the series, without any intervening doses.

[0154] Methods according to aspects of the present disclosure may include administering any number of secondary and / or tertiary doses to a patient. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) tertiary doses are administered to a patient.

[0155] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during treatment depending on the needs of the individual patient after clinical testing.

[0156] index In certain aspects, provided herein are methods of treating cancer using the CAR T cells provided herein. In certain embodiments, the cancer being treated expresses a PIG antigen and / or a low density antigen to which the CAR expressed on the T cell specifically binds.

[0157] In some embodiments, cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancer cells of the cervix, anus, vagina, vulva, penis, base of tongue, larynx, tonsils, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharyngeal, neck, ovary, prostate, skin, non-melanoma skin cancer (NMSC), cutaneous squamous cell carcinoma (SCC), stomach, testis, tongue, or uterus. In addition, cancers may include, specifically, the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; hairy cell carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combined hepatocellular and cholangiocarcinomas; trabecular-trabecular adenomas; adenoid cystic carcinomas; adenocarcinomas with adenomatous polyps; adenocarcinomas, familial polyposis coli; solid tumors; malignant carcinoid tumors; bronchiolo-alveolar adenocarcinomas; breast Head adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; Epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; ;Malignant thymoma;Malignant ovarian stromal tumor;Malignant theca tumor;Malignant granulosa cell tumor;Malignant androgen tumor;Sertoli cell tumor;Malignant Leydig cell tumor;Malignant lipid cell tumor;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Hemangiocytoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma ;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian thyroid tumor;choriocarcinoma;malignant mesonephroma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;malignant odontogenic tumor;ameloblastic odontosarcoma;malignant ameloblastoma;ameloblastic fibrosarcoma;malignant pinealoma;chordoma;malignant glioma;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrillary astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioblastoma;primitive neuroectodermal;cerebellar sarcoma;ganglionucleoblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;malignant meningioma;neurofibrosarcoma;malignant neurilemmoma;malignant granular cell tumor;malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma The tumor may be, but is not limited to, granuloma, small lymphocytic lymphoma, diffuse large cell lymphoma, follicular lymphoma, mycosis fungoides, other certain non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0158] In certain embodiments, the disclosed CAR-T cells can be used in combination with any compound, moiety, or group that has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents that may function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators, and are used in particular in cancer treatment. Exemplary anti-cancer compounds include alemtuzumab (Campath®), alitretinoin (Panretin®), anastrozole (Arimidex®), bevacizumab (Avastin®), bexarotene (Targretin®), bortezomib (Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), and rivaroxaban (Ricin®). Other notable cancers include rhabdomyolysis (Rabbitux®), ... (R), fulvestrant (Faslodex®), gefitinib (Iressa®), ibritumomab tiuxetan (Zevalin®), imatinib mesylate (Gleevec™), ipilimumab (Yervoy™), lapatinib ditosylate (Tykerb®), letrozole (Femara®), nilotinib (Tasigna®), ofatumumab (A rzerra®), panitumumab (Vectibix®), pazopanib hydrochloride (Votrient®), pertuzumab (Perjeta™), pralatrexate (Folotyn®), regorafenib (Stivarga®), rituximab (Rituxan®), romidepsin (Istodax®), sorafenib tosylate (Nexavar®),These include, but are not limited to, sunitinib malate (Sutent®), tamoxifen, temsirolimus (Torisel®), toremifene (Fareston®), tositumomab and 131I tositumomab (Bexxar™), trastuzumab (Herceptin®), tretinoin (Vesanoid®), vandetanib (Caprelsa®), vemurafenib (Zelboraf®), vorinostat (Zolinza®), and dib-aflibercept (Zaltrap®). Further examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycins). 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin,Specifically, calicheamicin gamma 1 and calicheamicin omega 1; dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; as well as the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, ca Mitomycins such as caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C. Mycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate ;Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;Androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;Aminoglutethimide, mitotane,Antiadrenal agents such as trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids and ansamitocins such as meitaisin; mitoguazone; mitoxantrone; mopidanol (mopidanmol);nitraerine;pentostatin;phenamet;pirarubicin;rosoxantrone;podophyllic acid;2-ethylhydrazide;procarbazine;PSK polysaccharide complex) and other antibiotics;razoxane;rhizoxin;sizofuran;spirogermanium;tenuazonic acid;triazicon;2,2´,2´´-trichlorotriethylamine;trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin);urethane;bi cytosine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacitosine;arabinoside ("Ara-C");cyclophosphamide;thiotepa;taxoids, such as paclitaxel and doxataxel;chlorambucil;gemcitabine;6-thioguanine;mercaptopurine;methotrexate;platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin;vinblastine;platinum;etoposide (VP-16);ifo Examples of chemotherapeutic agents include, but are not limited to, sufamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0159] In some embodiments, the subject is also administered an additional immunotherapeutic agent. Immunotherapy refers to treatments that use the subject's immune system to treat cancer, such as the use of cancer vaccines, cytokines, cancer-specific antibodies, T cell therapy, and dendritic cell therapy.

[0160] In some embodiments, the subject is also administered an immunomodulatory protein, examples of which include B-lymphocyte chemoattractant ("BLC"), CC motif chemokine 11 ("Eotaxin-1"), eosinophil chemotactic protein 2 ("Eotaxin-2"), granulocyte colony-stimulating factor ("G-CSF"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), 1-309, intercellular adhesion molecule 1 ("ICAM-1"), interferon gamma ("IFN-γ"), interleukin-1 alpha ("IL-1α"), interleukin-1 beta ("IL-1β"), interleukin 1 receptor, and the like. antagonist ("IL-1ra"), interleukin-2 ("IL-2"), interleukin-4 ("IL-4"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-6 soluble receptor ("IL-6sR"), interleukin-7 ("IL-7"), interleukin-8 ("IL-8"), interleukin-10 ("IL-10"), interleukin-11 ("IL-11"), interleukin-12 subunit beta ("IL-12p40" or "IL-1 2p70"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 ("IL-17"), chemokine (CC motif) ligand 2 ("MCP-1"), macrophage colony-stimulating factor ("M-CSF"), monokine induced by gamma interferon ("MIG"), chemokine (CC motif) ligand 2 ("MIP-1α"), chemokine (CC motif) ligand 4 ("MIP-1β"), macrophages. multi-inflammatory protein-1-delta ("MIP-1delta"), platelet-derived growth factor subunit B ("PDGF-BB"), chemokine (CC motif) ligand 5, regulated upon activation of conventional T cell expressed and secreted ("RANTES"), TIMP metallopeptidase inhibitor 1 ("TIMP-1"), TIMP metallopeptidase inhibitor 2 ("TIMP-2"), tumor necrosis factor, lymphotoxin alpha ("TNFα"), tumor necrosis factor, lymphotoxin beta ("TNFβ"), soluble TNF receptor type 1 ("sTNFRI");sTNFRIIAR, brain-derived neurotrophic factor ("BDNF"), basic fibroblast growth factor ("bFGF"), bone morphogenetic protein 4 ("BMP-4"), bone morphogenetic protein 5 ("BMP-5"), bone morphogenetic protein 7 ("BMP-7"), nerve growth factor ("b-NGF"), epidermal growth factor ("EGF"), epidermal growth factor receptor ("EGFR"), endocrine-derived vascular endothelial growth factor ("EG-VEGF"), fibroblast growth factor 4 ("FGF-4"), keratinocyte growth factor ("FGF-7"), growth differentiation factor 15 ("GDF-15"), glial cell line-derived neurotrophic factor ( "GDNF"), growth hormone, heparin-binding EGF-like growth factor ("HB-EGF"), hepatocyte growth factor ("HGF"), insulin-like growth factor binding protein 1 ("IGFBP-1"), insulin-like growth factor binding protein 2 ("IGFBP-2"), insulin-like growth factor binding protein 3 ("IGFBP-3"), insulin-like growth factor binding protein 4 ("IGFBP-4"), insulin-like growth factor binding protein 6 ("IGFBP-6"), insulin-like growth factor 1 ("IGF-1"), insulin, macrophage colony-stimulating factor ("M-CSF"), R"), nerve growth factor receptor ("NGF R"), neurotrophin-3 ("NT-3"), neurotrophin-4 ("NT-4"), osteoprotegerin ("osteoprotegerin"), platelet-derived growth factor receptor ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp, Cullin, F-box-containing complex ("SCF"), stem cell factor receptor ("SCF R"), transforming growth factor alpha ("TGFα"), transforming growth factor beta 1 ("TGFβ1"), transforming growth factor beta 3 ("TGFβ3"), vascular endothelial growth factor ("VEGF"), vascular endothelial growth factor receptor 2 ("VEGFR2"), vascular endothelial growth factor receptor 3 ("VEGFR3"), VEGF-D 6Ckine, tyrosine-protein kinase receptor UFO ("Axl"), Betacellulin ("BTC"), mucosa-associated epithelial chemokine ("CCL28"), chemokine (CC motif) ligand 27 ("CTACK"), chemokine (CXC motif) ligand 16 ("CXCL16");Chemokine with C-X-C motif 5 ("ENA-78"), chemokine (C-C motif) ligand 26 ("eotaxin-3"), granulocyte chemoattractant protein 2 ("GCP-2"), GRO, chemokine (C-C motif) ligand 14 ("HCC-1"), chemokine (C-C motif) ligand 16 ("HCC-4"), interleukin-9 ("IL-9"), interleukin-17F ("IL-17F"), interleukin-18 binding protein ("IL-18"), and IL-18 binding protein ("IL-18") have been reported. BPa"), interleukin-28A ("IL-28A"), interleukin-29 ("IL-29"), interleukin-31 ("IL-31"), C-X-C motif chemokine 10 ("IP-10"), chemokine receptor CXCR3 ("I-TAC"), leukemia inhibitory factor ("LIF"), Light, chemokine (C motif) ligand ("lymphotactin"), monocyte chemotactic protein 2 ("MCP-2"), monocyte chemotactic protein 3 ("MCP-3"), monocyte chemotactic protein 4 ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibitory factor ("MIF"); Chemokine (CC motif) ligand 20 ("MIP-3α"), CC motif chemokine 19 ("MIP-3β"), chemokine (CC motif) ligand 23 ("MPIF-1"), macrophage-stimulating protein alpha chain ("MSPα"), nucleosome assembly protein 1-like 4 ("NAP-2"), secreted phosphoprotein 1 ("osteopontin"), lung activity regulated cytokine ("PARC"), platelet factor 4 ("PF4"), stromal cell-derived factor 1 alpha ("SDF-1α"), chemokine (CC motif) ligand 17 ("TARC"), thymic expressed chemokine ("TECK"), thymic stromal lymphopoietin ("TSLP"), and thymic stromal lymphopoietin ("TSLP") are also included in this specification. 4- IBB"), CD 166 antigen ("ALCAM"), cluster of differentiation 80 ("B7-1"), tumor necrosis factor receptor superfamily member 17 ("BCMA"), cluster of differentiation 14 ("CD14"), cluster of differentiation 30 ("CD30"), cluster of differentiation 40 ("CD40 ligand"), carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) ("CEACAM-1"), death receptor 6 ("DR6"), deoxythymidine kinase ("Dtk"),type 1 membrane glycoprotein ("endoglin"), receptor tyrosine-protein kinase erbB-3 ("ErbB3"), endothelial leukocyte adhesion molecule 1 ("E-selectin"), apoptosis antigen 1 ("Fas"), Fms-like tyrosine kinase 3 ("Flt-3L"), tumor necrosis factor receptor superfamily member 1 ("GITR"), tumor necrosis factor receptor superfamily member 14 ("HVEM"), intercellular adhesion molecule 3 ("ICAM-3"), IL-1 R4, IL-1RI, IL-10Rβ, IL-17R, IL-2Rγ, IL-21R, lysosomal membrane protein 2 ("LIMPII"), neutrophil gelatinase-associated lipocalin ("lipocalin-2"), CD62L ("L-selectin"), lymphatic endothelial ("LYVE-1"), MHC class I polypeptide-related sequence A ("MICA"), MHC class I polypeptide-related sequence B ("MICB"), NRGl-βl, platelet-derived growth factor receptor beta ("PDGF Rβ"), platelet endothelial cell adhesion molecule ("PECAM-1"), RAGE, hepatitis A virus cell receptor 1 ("TIM-1"), tumor necrosis factor receptor superfamily member IOC ("TRAIL-1"), and / or IL-2R. R3"), Trappin protein transglutaminase binding domain ("Trappin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDAR, activin A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, cathepsin S, CD40, latent family protein IB ("Cripto-1"), DAN, Dickkopf-related protein 1 ("DKK-1"), E-cadherin, epithelial cell adhesion molecule (" "EpCAM"), Fas Ligand (FasL or CD95L), FcgRIIB / C, follistatin, galectin-7, intercellular adhesion molecule 2 ("ICAM-2"), IL-13Rl, IL-13R2, IL-17B, IL-2Ra, IL-2Rb, IL-23, LAP, neural cell adhesion molecule ("NrCAM"), plasminogen activator inhibitor 1 ("PAI-1"), platelet-derived growth factor receptor ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1β"), sgpl30, secreted frizzled-related protein 2 ("ShhN");Sialic acid-binding immunoglobulin-type lectin ("Siglec-5"), ST2, transforming growth factor beta 2 ("TGFβ2"), Tie-2, thrombopoietin ("TPO"), tumor necrosis factor receptor superfamily member 10D ("TRAIL R4"), triggering receptor expressed on myeloid cells 1 ("TREM-1"), vascular endothelial growth factor C ("VEGF-C"), VEGFRl, adiponectin, adipsin ("AND"), alpha-fetoprotein ("AFP"), angiopoietin-like 4 ("ANGPTL4"), beta-2-microglobulin ("B2M"), basal cell adhesion molecule ("BCAM"), carbohydrate antigen 125 ("CA125"), cancer antigen 15-3 ("CA15-3"), carcinoembryonic antigen ( "CEA"), cAMP receptor protein ("CRP"), human epidermal growth factor receptor 2 ("ErbB2"), follistatin, follicle maturation hormone ("FSH"), chemokine (CXC motif) ligand 1 ("GROα"), human chorionic gonadotropin ("βHCG"), insulin-like growth factor 1 receptor ("IGF-1sR"), IL-1sRII, IL-3, IL-18Rb, IL-21, leptin, matrix metalloproteinase-1 ("MMP-1") , matrix metalloproteinase-2 ("MMP-2"), matrix metalloproteinase-3 ("MMP-3"), matrix metalloproteinase-8 ("MMP-8"), matrix metalloproteinase-9 ("MMP-9"), matrix metalloproteinase-10 ("MMP-10"), matrix metalloproteinase-13 ("MMP-13"), neural cell adhesion molecule ("NCAM-1"), entactin ("nidogen-1"), and neuroprotection molecule ("NCAM-2"). specific enolase ("NSE"), oncostatin M ("OSM"), procalcitonin, prolactin, prostate-specific antigen ("PSA"), sialic acid-binding Ig-like lectin 9 ("Siglec-9"), ADAM17 endopeptidase ("TACE"), thyroglobulin, inhibitor of metalloproteinase 4 ("TIMP-4"), TSH2B4, a disintegrin and metalloproteinase domain-containing protein 9 ("ADAM-9"), angiopoietin 2,Tumor necrosis factor ligand superfamily member 13 / acidic leucine-rich nuclear phosphoprotein 32 family member B ("APRIL"), bone morphogenetic protein 2 ("BMP-2"), bone morphogenetic protein 9 ("BMP-9"), complement component 5a ("C5a"), cathepsin L, CD200, CD97, chemerin, tumor necrosis factor receptor superfamily member 6B ("DcR3"), fatty acid binding protein 2 ("FABP2"), fibroblast activation protein alpha ("F, AP"), fibroblast growth factor 19 ("FGF-19"), galectin-3, hepatocyte growth factor receptor ("HGF R"), IFN-α / βR2, insulin-like growth factor 2 ("IGF-2"), insulin-like growth factor 2 receptor ("IGF-2R"), interleukin-1 receptor 6 ("IL-1R6"), interleukin 24 ("IL-24"), interleukin 33 ("IL-33"), kallikrein 14, asparaginyl endopeptidase ("legumain"), oxidized low density lipoprotein receptor 1 ("LOX-1"), mannose-binding lectin ("MBL"), neprilysin ("NEP"), Notch homodimer Log 1, translocation associated (Drosophila) ("Notch-1"), nephroblastoma overexpression ("NOV"), osteoactivin, programmed cell death protein 1 ("PD-1"), N-acetylmuramoyl-L-alanine amidase ("PGRP-5"), serpin A4, secreted frizzled-related protein 3 ("sFRP-3"), thrombomodulin, Toll-like receptor 2 ("TLR2"), tumor necrosis factor receptor superfamily member 10A ("TRAIL"), and IL-16 ("IL-16"). Rl"), transferrin ("TRF"), WIF-lACE-2, albumin, AMICA, angiopoietin 4, B cell activating factor ("BAFF"), carbohydrate antigen 19-9 ("CA19-9"), CD 163, clusterin, CRT AM, chemokine (CXC motif) ligand 14 ("CXCL14"), cystatin C, decorin ("DCN"), Dickkopf related protein 3 ("Dkk-3"), delta-like protein 1 ("DLL1"), fetuin A, heparin-binding growth factor 1 ("aFGF"), folate receptor alpha ("FOLR1"), furin, GPCR-associated sorting protein 1 ("GASP-1"), GPCR-associated sorting protein 2 ("GASP-2"), granulocyte-colony stimulating factor receptor ("GCSF"). R"), serine protease hepsin ("HAI-2"), interleukin-17B receptor ("IL-17B R"), interleukin 27 ("IL-27"), lymphocyte activation gene 3 ("LAG-3"), apolipoprotein AV ("LDLThese include, but are not limited to, VE-cadherin (vascular endothelial), also known as cadherin 5, type 2 or CD144 ("VE-cadherin"), WNT1-inducible signaling pathway protein 1 ("WISP-1"), and receptor activator of nuclear factor of kappa B ("RANK").

[0161] The disclosed CARs and immune cells expressing the CARs can be used in combination with immune checkpoint inhibitors. Immune checkpoint inhibition broadly refers to inhibiting checkpoints that cancer cells may initiate to prevent or downregulate an immune response. Two known immune checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play key roles at all stages of T-cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the primary ligand, while PD-L2 has a much more restricted expression pattern. Binding of ligand to PD-1 delivers an inhibitory signal to T cells, thereby reducing cytokine production and suppressing T-cell proliferation. Checkpoint inhibitors include, but are not limited to, aptamers and antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475, AMP-514), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7, AMP-224), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).Immune checkpoint inhibitors include cemiplimab (REGN2810), nivolumab (BMS-936558, MDX-1106, ONO-4538), pembrolizumab (MK-3475, SCH 900475), atezolizumab (MPDL3280A, RG7446, RO5541267), durvalumab (MEDI4736, MEDI-4736), avelumab (MSB0010718C), ipilimumab (BMS-734016, IBI310, MDX-010), SHR1210, sintilimab (IBI308), spartalizumab (PDR001), surelizumab (BGB-A317), pidilizumab, BCD-100, toripalimab (JS001), BAY 1905254, ASP 8374, PF-06801591, AMP-224, AB122, AK105, AMG 404, BCD-100, BI 754091, F520, HLX10, HX008, JTX-4014, LZM009, MEDI0680, MGA012, Sym021, TSR-042, PSB205, MGD019, MGD013, AK104, XmAb20717, RO71216 61, CX-188, INCB086550, FS118, BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, HLX20, KN035, MDX-1105, MSB2311, SHR-1316, TG-15 01, ZKAB001, INBRX-105, MCLA-145, KN046, M7824, LY3415244, INCB086550, CA-170, CX-072, ADU-1604, AGEN1181, AGEN1884, MK-1308, REGN4659, XmAb22841, ATOR-1015, PSB205, MGD019, AK104, XmAb20717, BMS-986249, tremelimumab, BMS-986258, BGB-A425, INCAGN02390, Sym023, JNJ 61610588, BI 754111, LAG525, MK-4280, REGN3767, Sym022, TSR-033, leratolimab, JTX-2011, MGD009, BMS-986207, OMP-313M32, MK-7684 or TSR-022.

[0162] Human monoclonal antibodies against programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapies are described in U.S. Patent No. 8,008,449, which are incorporated by reference. Anti-PD-L1 antibodies and their uses are described in U.S. Patent No. 8,552,154, which are incorporated by reference. Anti-cancer agents comprising anti-PD-1 or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which are incorporated by reference.

[0163] In some embodiments, the PD-L1 inhibitor comprises an antibody that specifically binds to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD-1 inhibitor comprises an antibody that specifically binds to PD-1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies against PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapies are described in U.S. Pat. No. 8,008,449, which are incorporated by reference. Anti-PD-L1 antibodies and their uses are described in U.S. Pat. No. 8,552,154, which are incorporated by reference. Anti-PD-1 antibodies or anti-cancer agents comprising anti-PD-L1 antibodies are described in U.S. Pat. No. 8,617,546, which are incorporated by reference.

[0164] In addition to T cell receptor activation, optimal "killer" CD8 T cell responses require costimulation, which can be achieved by ligation of tumor necrosis factor receptor family members including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because administration of activating (agonist) anti-OX40 mAbs enhances T cell differentiation and cytolysis, leading to enhanced antitumor immunity against a variety of tumors.

[0165] In some embodiments, such additional therapeutic agents may be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0166] In some embodiments, such additional therapeutic agents may be selected from alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin.

[0167] In some embodiments, such additional therapeutic agents can be selected from antimitotic agents such as taxanes, eg, docetaxel, and paclitaxel, and vinca alkaloids, eg, vindesine, vincristine, vinblastine, and vinorelbine.

[0168] In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors, such as topotecan or irinotecan, or cytostatic agents, such as etoposide and teniposide.

[0169] In some embodiments, such additional therapeutic agents may be selected from a growth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such as an EGFR antibody, e.g., zalutumumab, cetuximab, panitumumab, or nimotuzumab, or other EGFR inhibitors such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as a HER2 antibody, e.g., trastuzumab, trastuzumab-DM1, or pertuzumab), or an inhibitor of both EGFR and HER2 (such as lapatinib).

[0170] In some embodiments, such additional therapeutic agents may be selected from tyrosine kinase inhibitors such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0171] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0172] In some embodiments, the therapeutic agent used in combination with the CARs (e.g., CARs disclosed herein and immune cells expressing such CARs) to treat the aforementioned disorders may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR) negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0173] In some embodiments, the therapeutic agent used in combination with the CAR to treat the aforementioned disorders may be a cell cycle control / apoptosis regulator (or "regulator"). Cell cycle control / apoptosis regulators may include molecules that target and regulate cell cycle control / apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modifiers (such as BIBR1532, SOT-095, GRN163, and compositions described in, for example, US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0174] In some embodiments, therapeutic agents used in combination with CARs (e.g., CARs disclosed herein and immune cells expressing such CARs) to treat the aforementioned disorders can be hormone modulating agents, such as antiandrogens and agents useful in estrogen therapy. Examples of such hormone modulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, antiandrogens (such as flutamide / eulexin), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone / provera, megestrol acepate / megace, etc.), corticosteroids (such as hydrocortisone, prednisone), luteinizing hormone releasing hormone (and its analogs, as well as other LHRH agonists such as buserelin and goserelin), aromatase inhibitors (such as anastrozole / arimidex, aminoglutethimide / cytraden, exemestane, etc.), or hormone inhibitors (such as octreotide / sandostatin).

[0175] As mentioned above, the co-administration may be simultaneous, separate or sequential. In the case of simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0176] Working Example Example 1: SK-MEL-37 tumor and anti-HLA-A2 / MAGEA4 286-294 Chimeric antigen receptors containing scFv Preparation of CAR: V L -V H Directional anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs.286-294 V of antibody 31345 L and V H The BB / z CARs were constructed using the sequence. As a non-binding control, BB / z CARs were designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to the different scFvs. These CARs were cloned into pLVX lentiviral vectors containing EF1a promoter and IRES:eGFP sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses. See Figure 1 part B for construct schematic.

[0177] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors ("Donor 1" and "Donor 2"), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for 19 days and then cryopreserved until use in in vivo experiments.

[0178] Xenograft and measurement of tumor xenografts HLA-A2 / MAGEA4 286-294 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + MAGEA4 + SK-MEL-37 human melanoma tumor cells were injected subcutaneously. SK-MEL-37 melanoma cells express MAGEA4 286-294 Mass spectrometry was used to confirm that the mice expressed approximately 1,326 cell surface copies of the peptide. On day 7 after tumor establishment, mice (n=5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 CAR, or a combination of both CARs from two different donors. 286-294 BB / z CAR or anti-HLA-A2 / MAGEA4286-294 4 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed up to day 64 by measuring tumor volume.

[0179] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0180] In summary, the results show that MAGEA4 286-294 28 / z CAR T cells are 286-294 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0181] [Table 10]

[0182] Experimental Procedure: Summary of Results and Conclusions: Donor 1: SK-MEL-37 tumors were treated with control CAR T cells or MAGEA4 286-294 BB / z CAR T cells grew progressively in mice that received either 286-294 28 / z suppressed the growth of established SK-MEL-37 tumors in vivo. 286-294 MAGEA4 in contrast to BB / z CAR 286-294 The enhanced efficacy of 28 / z CAR was confirmed with statistically significant differences in tumor size at days 31, 35, 40, 47, 55, and 62 with p<0.0001 by 2-way ANOVA test. See Figure 2, part A.

[0183] Donor 2: SK-MEL-37 tumors grew progressively in mice receiving control CAR T cells. MAGEA4 286-294 Administration of BB / z CAR T cells was effective, slowing tumor growth by approximately one week. However, MAGEA4 286-294 28 / z CAR T cells potently suppressed SK-MEL-37 tumor growth, rendering tumors undetectable (not palpable) in 5 of 5 mice by day 20. These tumors remained undetectable until days 62–69, when tumors recurred. 286-294 MAGEA4 in contrast to BB / z CAR 286-294 The enhanced antitumor effect of 28 / z CAR was confirmed, with tumor size at day 35 being statistically significant by two-way ANOVA test (p=0.0037), and tumor sizes at days 40, 47, 55, and 62 also being statistically significant with p<0.0001. See Figure 2, part B.

[0184] [Table 11-1]

[0185] [Table 11-2]

[0186] [Table 11-3]

[0187] [Table 11-4]

[0188] [Table 11-5]

[0189] [Table 11-6]

[0190] [Table 11-7]

[0191] [Table 11-8]

[0192] [Table 11-9]

[0193] Example 2: A375 tumor and anti-HLA-A2 / MAGEA4 286-294 Chimeric antigen receptors containing scFv Constructs and CAR T cells V L -V H Directional anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 286-294 V of antibody 31345 L and V H The BB / z CARs were constructed using the scFv sequence. As a non-binding control, the BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to the scFv. These CARs were cloned into the pLVX lentiviral vector containing the EF1a promoter and IRES:eGFP sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figure 1, part B for construct schematic and Figure 3 for results.

[0194] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors ("Donor 1" and "Donor 2"), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for 19 days and then cryopreserved until use in in vivo experiments.

[0195] Xenograft and measurement of tumor xenografts HLA-A2 / MAGEA4 286-294 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. A375 melanoma cells express MAGEA4 286-294 Mass spectrometry was used to confirm that the expression of approximately 424 cell surface copies of the peptide was achieved. On day 13 after tumor establishment, mice (n=5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 CAR, or a combination of both CARs from two different donors. 286-294 BB / z CAR or anti-HLA-A2 / MAGEA4 286-294 4 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed up to day 64 by measuring tumor volume.

[0196] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2) / 2.

[0197] Summary of results and conclusions: Donor 1: A375 tumors received control CAR T cells or MAGEA4 286-294 BB / z CAR T cells grew progressively in mice that received either 286-294 28 / z inhibited the growth of established A375 tumors in vivo, with 1 in 5 mice being tumor-free at day 64. 286-294 MAGEA4 in contrast to BB / z CAR 286-294 The enhanced efficacy of 28 / z CAR was confirmed with statistically significant differences in tumor size at days 27, 29, 33, 36, 40, and 44 with p<0.0001 by 2-way ANOVA test. See Figure 3, part A.

[0198] Donor 2: A375 tumors grew progressively in mice receiving control CAR T cells. MAGEA4 286-294 BB / z and MAGEA4 286-294 Both administrations of 28 / z CAR T cells suppressed A375 tumor growth, but with different kinetics. 286-294 28 / z CAR T cells acted with faster kinetics, with tumors disappearing in 5 out of 5 mice by day 27. 286-294 BB / z CAR T cells acted with slower kinetics, with tumors disappearing in 4 out of 5 mice by day 44. 286-294 MAGEA4 in contrast to BB / z CAR 286-294 The enhanced kinetics of the antitumor effect of 28 / z CAR was confirmed, with the tumor sizes at days 19 and 22 being statistically significant at p=0.0071 and p=0.0008, respectively, by two-way ANOVA test. See Figure 3, part B.

[0199] [Table 12-1]

[0200] [Table 12-2]

[0201] [Table 12-3]

[0202] [Table 12-4]

[0203] [Table 12-5]

[0204] [Table 12-6]

[0205] [Table 12-7]

[0206] [Table 12-8]

[0207] In summary, the results of Examples 1 and 2 show that MAGEA4 286-294 28 / z CAR T cells are 286-294 It was revealed that BB / z CAR T cells exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells (Figure 2, Parts A and B, and Figure 3, Parts A and B).

[0208] Example 3: Anti-HLA-A2 / MAGEA4 230-239 Chimeric antigen receptors containing scFv V L -V H Directional anti-HLA-A2 / MAGEA4 230-239In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 33229P L and V H The 28 / z CAR was constructed using the sequence. As a non-binding control, the 28 / z CAR was designed using the huCD28 hinge / transmembrane / co-stimulatory domain and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into the pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figure 4, part B for the constructs and Figure 4, part A for the results.

[0209] [Table 13]

[0210] [Table 14]

[0211] Experimental procedure Generation of CAR constructs and CAR T cells V L -V H Directional anti-HLA-A2 / MAGEA4 230-239 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 33229P L and V HAs a non-binding control, 28 / z CARs were designed using the huCD28 hinge / transmembrane / co-stimulatory domains and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses.

[0212] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of a standard donor, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for approximately 14 days and then cryopreserved until use in in vivo experiments.

[0213] Xenograft and measurement of tumor xenografts Anti-HLA-A2 / MAGEA4 230-239 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells directed against immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. A375 melanoma cells express HLA-A2 / MAGEA4 230-239 Mass spectrometry was used to confirm that approximately 553 cell surface copies of the peptide were expressed. On day 13 after tumor establishment, mice (n=4 or 5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 4 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing either GFP, a marker for CAR-transduced cells) 6T cells were injected intravenously. Tumor growth was assessed up to day 28 by measuring tumor volume.

[0214] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0215] Summary of results and conclusions: A375 tumors grew progressively in untreated mice and in mice treated with control CAR T cells. 230-239 Mice receiving BB / z CAR T cells showed some tumor control with reduced tumor growth on days 19 (p<0.02), 23 (p<0.02), and 26 (p<0.0001) compared to mice receiving control CAR T (analyzed statistically by two-way ANOVA). 230-239 Administration of 28 / z CAR T also resulted in inhibition of established A375 tumor growth on days 19 (p=0.007), 23 (p<0.0001), and 26 (p<0.0001) (statistically analyzed by two-way ANOVA). 230-239 Anti-HLA-A2 / MAGEA4 versus BB / z CAR 230-239 The enhanced efficacy of 28 / z CAR was confirmed with tumor size statistically significant at day 23 (p<0.0001) and day 26 (p<0.0001) by 2-way ANOVA. See Figure 4, part A.

[0216] In summary, the results show that anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells are anti-HLA-A2 / MAGEA4 230-239 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0217] [Table 15-1]

[0218] [Table 15-2]

[0219] [Table 15-3]

[0220] Example 4: Anti-HLA-A2 / Tyrosinase 369-377 Chimeric antigen receptors containing scFv V L -V H Directional anti-HLA-A2 / tyrosinase 369-377 In addition to the scFv, a chimeric antigen receptor containing either 1) a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain (BB / z CAR) or 2) a huCD28 hinge / transmembrane / costimulatory domain, and a CD3z signaling domain (28 / z CAR) was used as an anti-HLA-A2 / tyrosinase 369-377 V of antibody D11 (identified by patent WO / 2016 / 199141) L and V H The sequence was constructed using: Anti-HLA-A2 / tyrosinase 369-377 The antibody is HLA-A2 / tyrosinase 369-377 Similarly, HLA-A2 / tyrosinase in which N371 is deamidated to D371 369-377(N371D) As a non-binding control, BB / z CARs were designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses. See Figure 5 part A for construct schematic.

[0221] In summary, the results show that anti-HLA-A2 / tyrosinase antibodies from two donors 369-377 28 / z CAR T cells are anti-HLA-A2 / tyrosinase 369-377 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0222] [Table 16]

[0223] [Table 17]

[0224] Experimental procedure Generation of CAR constructs and CAR T cells V L -V H Directional anti-HLA-A2 / tyrosinase 369-377 In addition to the scFv, a chimeric antigen receptor containing either 1) a huCD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3z signaling domain (BB / z CAR) or 2) a huCD28 hinge / transmembrane / costimulatory domain, and a CD3z signaling domain (28 / z CAR) was used as an anti-HLA-A2 / tyrosinase 369-377 V of antibody D11 (identified by patent WO / 2016 / 199141) L and V H The BB / z CARs were constructed using the sequence. As a non-binding control, the BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into the pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figure 5 part A for a schematic of the construct.

[0225] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors ("Donor 1" and "Donor 2"), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for 19 days and then cryopreserved until use in in vivo experiments.

[0226] Xenograft and measurement of tumor xenografts Anti-HLA-A2 / tyrosinase 369-377 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells directed against immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 4 × 10 6 HLA-A2 + Tyrosinase + WM266.4 human melanoma tumor cells were injected subcutaneously. WM266.4 melanoma cells express HLA-A2 / tyrosinase 369-377 Mass spectrometry was used to confirm that the peptide expressed approximately 4,500 cell surface copies. On day 7 after tumor establishment, mice (n=5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / tyrosinase CAR T, or a combination of both from two different donors. 369-377 BB / z CAR or anti-HLA-A2 / tyrosinase 369-377 2 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed up to day 90 by measuring tumor volume.

[0227] A second xenogeneic tumor study was performed using a different tumor cell line. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 +Tyrosinase + SK-MEL-23 human melanoma tumor cells were injected subcutaneously. SK-MEL-23 melanoma cells express HLA-A2 / tyrosinase 369-377 Mass spectrometry was used to confirm that the peptide expressed approximately 2,000-3,000 cell surface copies. On day 7 after tumor establishment, mice (n=5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / tyrosinase CAR T, or a combination of both from two different donors. 369-377 BB / z CAR or anti-HLA-A2 / tyrosinase 369-377 4 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed up to day 90 by measuring tumor volume.

[0228] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0229] Summary of results and conclusions: WM266.4 Tumor Model: Donor 1: WM266.4 tumors grew progressively in mice not receiving any therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / tyrosinase 369-377 Mice receiving BB / z CAR T cells showed some efficacy compared to controls, with reduced tumor growth on days 31 (p=0.02), 35 (p=0.001), 42 (p=0.0004), and 49 (p<0.0001) (statistically analyzed by two-way ANOVA). In contrast, anti-HLA-A2 / tyrosinase 369-377 Administration of 28 / z CAR T resulted in regression of established WM266.4 tumors, with 4 of 5 mice remaining tumor-free by day 90. Anti-HLA-A2 / tyrosinase 369-377Anti-HLA-A2 / tyrosinase versus BB / z CAR 369-377 The enhanced efficacy of 28 / z CAR was confirmed with tumor sizes at days 35, 42, and 49 being statistically significant by 2-way ANOVA (p=0.01, p<0.0001, and p<0.000, respectively). See Figure 5, part B.

[0230] Donor 2: WM266.4 tumors grew progressively in mice not receiving any therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / tyrosinase 369-377 Mice receiving BB / z CAR T cells showed some efficacy compared to controls, with reduced tumor growth on days 28 (p=0.03), 31 (p=0.004), 35 (p=0.0001), 42 (p=0.0001), and 49 (p<0.0001) (analyzed statistically by two-way ANOVA). In contrast, anti-HLA-A2 / tyrosinase 369-377 Administration of 28 / z CAR T resulted in regression of established WM266.4 tumors, with 5 of 5 mice remaining tumor-free by day 90. Anti-HLA-A2 / tyrosinase 369-377 Anti-HLA-A2 / tyrosinase versus BB / z CAR 369-377 The enhanced efficacy of 28 / z CAR was confirmed with tumor sizes on days 24, 28, 31, 35, 42, and 49 statistically significant by 2-way ANOVA (p=0.03, p=0.008, p=0.0004, p<0.0001, p<0.0001, and p<0.0001, respectively). See Figure 5, part B.

[0231] SK-MEL-23 tumor model: Donor 1: SK-MEL-23 tumors grew progressively in mice not receiving any therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / tyrosinase 369-377 Mice that received BB / z CAR T cells also showed progressive tumor growth similar to controls. In contrast, anti-HLA-A2 / tyrosinase 369-377Treatment with 28 / z CAR T significantly inhibited established SK-MEL-23 tumor growth on days 31 (p=0.02), 35 (p=0.004), 42 (p=0.0003), and on days 49, 55, 63, 72, 78, and 90 (p<0.0001 for these days) compared to animals receiving control CAR T cells (statistically analyzed by 2-way ANOVA). Anti-HLA-A2 / tyrosinase 369-377 Anti-HLA-A2 / tyrosinase versus BB / z CAR 369-377 The enhanced efficacy of 28 / z CAR was confirmed, with tumor sizes on days 35, 42, 49, 55, 63, 72, 78, and 90 being statistically significant by 2-way ANOVA test (p=0.04 and p=0.004 on days 35 and 42; p<0.0001 on all other days). See Figure 5, part C.

[0232] Donor 2: SK-MEL-23 tumors grew progressively in mice not receiving any therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / tyrosinase 369-377 Mice that received BB / z CAR T cells also showed progressive tumor growth similar to controls. In contrast, anti-HLA-A2 / tyrosinase 369-377 Administration of 28 / z CAR T resulted in a reduction in the size of established SK-MEL-23 tumors and inhibition of tumor growth on days 21 (p=0.02), 24 (p=0.004), and 28 (p=0.0004), as well as days 31, 35, 42, 49, 55, 63, 72, 78, and 90 (all p<0.0001 for these days) compared to animals receiving control CAR T cells (statistically analyzed by 2-way ANOVA). 369-377 Anti-HLA-A2 / tyrosinase versus BB / z CAR 369-377The enhanced efficacy of 28 / z CAR was confirmed with tumor sizes on days 21, 24, 28, 31, 35, 42, 49, 55, 63, 72, 78, and 90 being statistically significant by 2-way ANOVA test (p=0.01 and p=0.005 for days 21 and 24; p<0.0001 for all other days).

[0233] [Table 18-1]

[0234] [Table 18-2]

[0235] [Table 18-3]

[0236] [Table 18-4]

[0237] [Table 18-5]

[0238] [Table 18-6]

[0239] [Table 18-7]

[0240] [Table 18-8]

[0241] [Table 18-9]

[0242] [Table 18-10]

[0243] [Table 18-11]

[0244] [Table 18-12]

[0245] [Table 18-13]

[0246] [Table 18-14]

[0247] [Table 18-15]

[0248] [Table 18-16]

[0249] Example 5: Anti-HLA-A2 / NY-ESO-1 157-165 Chimeric antigen receptors containing scFv V H -V L Directional anti-HLA-A2 / NY-ESO-1 157-165In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were co-expressed with anti-HLA-A2 / NY-ESO-1 157-165 V of antibody 28105P L and V H As a non-binding control, BB / z CARs were designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses. See Figure 6 for construct design (part A) and results (part B). In summary, the results demonstrate that anti-HLA-A2 / NY-ESO-1 CARs express the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. 157-165 28 / z CAR T cells are anti-HLA-A2 / NY-ESO-1 157-165 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0250] [Table 19]

[0251] [Table 20]

[0252] Testing Procedure: Generation of CAR constructs and CAR T cells V H -V L Directional anti-HLA-A2 / NY-ESO-1 157-165In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were co-expressed with anti-HLA-A2 / NY-ESO-1 157-165 V of antibody 28105P L and V H As a non-binding control, the BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses.

[0253] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of standard donors, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for 19 days and then cryopreserved until use in in vivo experiments.

[0254] Xenograft and measurement of tumor xenografts Anti-HLA-A2 / NY-ESO-1 157-165 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells directed against immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + NY-ESO-1 + A375 human melanoma tumor cells were injected subcutaneously. Three days after tumor establishment, mice (n=5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / NY-ESO-1 CAR, or a combination of both from two different donors.157-165 BB / z CAR or anti-HLA-A2 / NY-ESO-1 157-165 20 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed up to day 38 by measuring tumor volume.

[0255] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0256] Summary of results and conclusions: A375 tumors grew progressively in mice treated with control CAR T cells. Anti-HLA-A2 / NY-ESO-1 157-165 Mice receiving BB / z CAR T cells showed some tumor control with reduced tumor growth on days 21 (p<0.04), 28 (p<0.0001), and 38 (p<0.0001) compared to mice receiving control CAR T (analyzed statistically by two-way ANOVA). 157-165 Administration of 28 / z CAR T also resulted in inhibition of established A375 tumor growth on days 16 (p=0.03), 19 (p=0.0002), 21 (p<0.0001), 28 (p<0.0001), and 38 (p<0.0001) (statistically analyzed by two-way ANOVA). 157-165 Anti-HLA-A2 / NY-ESO-1 versus BB / z CAR 157-165 The enhanced efficacy of 28 / z CAR was confirmed with tumor size statistically significant at day 21 (p=0.002), day 28 (p<0.0001) and day 38 (p<0.0001) by 2-way ANOVA (p<0.0001 for both days). See Figure 6, part B.

[0257] [Table 21-1]

[0258] [Table 21-2]

[0259] [Table 21-3]

[0260] [Table 21-4]

[0261] Example 6: Dual anti-HLA-A2 / MAGEA4 230-239 One of the scFvs or anti-HLA-A2 / MAGEA4 286-294 In vitro cytotoxicity by T cells expressing chimeric antigen receptors containing Two anti-HLA-A2 / MAGEA4 230-239 scFv or anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) were co-expressed with two anti-HLA-A2 / MAGEA4 CARs. 230-239 Antibody (33229 or 34852) and anti-HLA-A2 / MAGEA4 286-294 V of antibody (31345) L and V HAs a non-binding control, BB / z CAR was designed using huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv, and 28 / z CAR was designed using huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vector containing EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figures 8 and 11 for construct design and Figure 7 for results.

[0262] [Table 22-1]

[0263] [Table 22-2]

[0264] Experimental procedure Generation of CAR constructs and CAR T cells Two anti-HLA-A2 / MAGEA4 230-239 scFv or anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) were co-expressed with two anti-HLA-A2 / MAGEA4 CARs. 230-239 Antibody (33229 or 34852) and anti-HLA-A2 / MAGEA4 286-294 V of antibody (31345) L and V HAs a non-binding control, the BB / z CAR was designed with an irrelevant scFv plus the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain, and the 28 / z CAR was designed with an irrelevant scFv plus the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain. These CARs were cloned into the pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses.

[0265] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of standard donors, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for 14 days and then cryopreserved until use in in vivo cytotoxicity assays.

[0266] Calcein release cytotoxicity assay Anti-HLA-A2 / MAGEA4 containing either the BB / z or 28 / z intracellular signaling domain 230-239 and anti-HLA-A2 / MAGEA4 286-294 A 2.5-h calcein release assay was performed to measure the in vitro cytotoxic activity of chimeric antigen receptor (CAR) T cells directed against A375 or SK-MEL-37 cells (2 × 10 6Cells / mL) were labeled with 8 μm Calcein-AM (ThermoFisher) for 35 min at 37°C and washed three times with medium. CAR-transduced activated / expanded T cells and calcein-labeled target cells were co-cultured at various ratios in triplicates in 96-well round-bottom plates for 2.5 h at 37°C. After incubation, culture supernatants were transferred to black clear-bottom 96-well microplates (Greiner Bio One) and calcein fluorescence was measured using a microplate reader. The percentage of calcein release was calculated as ((calcein signal-spontaneous calcein release) / (calcein maximum release-spontaneous calcein release))*100 and recorded as the percentage of cytotoxic effect. Spontaneous release was measured in target cells alone and maximum release was measured by lysing target cells with 1% Triton X-114 detergent.

[0267] Summary of results and conclusions MAGEA4 + HLA-A2 + Some cytotoxicity in A375 and SK-MEL-37 cells was observed with anti-MAGEA4 antibodies containing the BB / z intracellular signaling domain. 286-294 In contrast to the CAR (31345), anti-MAGEA4 contains the 28 / z intracellular signaling domain. 286-294 Enhanced cytotoxicity was observed with CAR (31345) (Figure 7). Minimal background cytotoxicity was induced by the CTL CAR construct (Figure 7). Thus, anti-MAGEA4 containing the 28 / z signaling domain 286-294 The CAR(31345) CAR induced superior cytotoxic effects compared to the same CAR utilizing the BB / z signaling domain.

[0268] MAGEA4 + HLA-A2 + Some cytotoxicity in A375 and SK-MEL-37 cells was observed with anti-MAGEA4 antibodies containing the BB / z intracellular signaling domain. 230-239 In contrast to the CAR (33229), anti-MAGEA4 contains the 28 / z intracellular signaling domain.239-239 CAR (33229) demonstrated enhanced cytotoxicity (Figure 7) and minimal background cytotoxicity was induced by the CTL CAR construct (Figure 7). Thus, anti-MAGEA4 containing the 28 / z signaling domain 230-239 The CAR(33229) CAR induced superior cytotoxic effects compared to the same CAR utilizing the BB / z signaling domain.

[0269] MAGEA4 + HLA-A2 + Some cytotoxicity in A375 cells was observed with anti-MAGEA4 antibodies containing the BB / z intracellular signaling domain. 230-239 In contrast to the CAR (34852), anti-MAGEA4 contains the 28 / z intracellular signaling domain. 239-239 Enhanced cytotoxicity was observed with CAR(34852) (Figure 7). Minimal background cytotoxicity was induced by the CTL CAR construct (Figure 7). Thus, anti-MAGEA4 containing the 28 / z signaling domain 230-239 The CAR(34852) CAR induced superior cytotoxic effects compared to the same CAR utilizing the BB / z signaling domain.

[0270] [Table 23-1]

[0271] [Table 23-2]

[0272] [Table 23-3]

[0273] [Table 23-4]

[0274] [Table 23-5]

[0275] [Table 23-6]

[0276] [Table 23-7]

[0277] [Table 23-8]

[0278] [Table 23-9]

[0279] Example 7: Dual anti-HLA-A2 / MAGEA4 230-239 Chimeric antigen receptor containing one of the scFvs V L -V H Directional anti-HLA-A2 / MAGEA4 230-239 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 33229P L and V HAs a non-binding control, 28 / z CAR was designed using the huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figures 9 and 10 for results and construct design, respectively.

[0280] In summary, the results show that anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells are anti-HLA-A2 / MAGEA4 230-239 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0281] [Table 24]

[0282] Experimental procedure Generation of CAR constructs and CAR T cells V L -V H Directional anti-HLA-A2 / MAGEA4 230-239 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 33229P L and V HAs a non-binding control, 28 / z CARs were designed using the huCD28 hinge / transmembrane / co-stimulatory domains and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses.

[0283] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of a standard donor, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for approximately 14 days and then cryopreserved until use in in vivo experiments.

[0284] Xenograft and measurement of tumor xenografts Anti-HLA-A2 / MAGEA4 230-239 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells directed against immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. A375 melanoma cells express HLA-A2 / MAGEA4 230-239 Mass spectrometry was used to confirm that approximately 553 cell surface copies of the peptide were expressed. On day 11 after tumor establishment, mice (n≧5 per group) were injected with either a non-binding control 28 / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 1 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing either GFP, a marker for CAR-transduced cells) 6 pieces, 5×10 6Pieces, or 10x10 6 T cells were injected intravenously. Tumor growth was assessed up to day 28 by measuring tumor volume.

[0285] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0286] Summary of results 1×10 6 Dose of CAR T:A375 tumors grew progressively in mice receiving control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239 Mice receiving BB / z CAR T cells did not show tumor control. In contrast, anti-HLA-A2 / MAGEA4 230-239 Administration of 28 / z CAR T resulted in inhibition of established A375 tumor growth on days 9 (p=0.001), 13 (p<0.0001), and 16 (p<0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). 230-239 Anti-HLA-A2 / MAGEA4 versus BB / z CAR 230-239 Enhanced efficacy of 28 / z CAR was confirmed with tumor size statistically significant at days 9 (p=0.0086), 13 (p<0.0001), and 16 (p<0.0001) by two-way ANOVA.

[0287] 5×10 6 Dose of CAR T:A375 tumors grew progressively in mice receiving control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239Administration of BB / z CAR T cells resulted in inhibition of established A375 tumor growth on days 9 (p<0.0001), 13 (p<0.0001), and 16 (p<0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). 230-239 Administration of 28 / z CAR T also resulted in inhibition of established A375 tumor growth on days 6 (p<0.0001), 9 (p<0.0001), 13 (p<0.0001), and 16 (p<0.0001) (statistically analyzed by two-way ANOVA). 230-239 Anti-HLA-A2 / MAGEA4 versus BB / z CAR 230-239 Enhanced efficacy of 28 / z CAR was confirmed with tumor size statistically significant at days 6 (p=0.0049), 9 (p<0.0001), 13 (p=0.0003), and 16 (p=0.0453) by two-way ANOVA.

[0288] 10×10 6 Dose of CAR T:A375 tumors grew progressively in mice receiving control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239 Administration of BB / z CAR T cells resulted in inhibition of established A375 tumor growth on days 6 (p=0.0214), 9 (p<0.0001), 13 (p<0.0001), and 16 (p<0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). 230-239 Administration of 28 / z CAR T also resulted in inhibition of established A375 tumor growth on days 6 (p<0.0001), 9 (p<0.0001), 13 (p<0.0001), and 16 (p<0.0001) (statistically analyzed by two-way ANOVA). 230-239 Anti-HLA-A2 / MAGEA4 versus BB / z CAR 230-239The enhanced efficacy of 28 / z CAR was confirmed with tumor size statistically significant at day 6 (p=0.0282) and day 9 (p=0.0212) by two-way ANOVA.

[0289] [Table 25-1]

[0290] [Table 25-2]

[0291] [Table 25-3]

[0292] [Table 25-4]

[0293] [Table 25-5]

[0294] [Table 25-6]

[0295] Example 8: Anti-HLA-A2 / MAGEA4 230-239 Chimeric antigen receptors containing scFv V L -V H Directional anti-HLA-A2 / MAGEA4 230-239 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 34852L and V H As a non-binding control, 28 / z CAR was designed using the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentivirus. See Figures 11 and 12 for construct design and results, respectively.

[0296] In summary, the results show that anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells are anti-HLA-A2 / MAGEA4 230-239 It was found that it exhibited superior in vivo antitumor activity and antitumor kinetics compared to BB / z CAR T cells.

[0297] [Table 26]

[0298] Experimental procedure Generation of CAR constructs and CAR T cells V L -V H Directional anti-HLA-A2 / MAGEA4 230-239 In addition to the scFv, chimeric antigen receptors containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain (28 / z CAR) were used as anti-HLA-A2 / MAGEA4 CARs. 230-239 V of antibody 34852 L and V HAs a non-binding control, 28 / z CARs were designed using the huCD28 hinge / transmembrane / co-stimulatory domains and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into pLVX lentiviral vectors containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses.

[0299] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of a standard donor, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads for approximately 14 days and then cryopreserved until use in in vivo experiments.

[0300] Xenograft and measurement of tumor xenografts Anti-HLA-A2 / MAGEA4 230-239 A xenogeneic tumor study was performed to measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells directed against immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. A375 melanoma cells express HLA-A2 / MAGEA4 230-239 Mass spectrometry was used to confirm that the mice expressed approximately 553 cell surface copies of the peptide. Ten days after tumor establishment, mice (n=5 per group) were injected with either a non-binding control 28 / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 CAR, or a combination of both CARs from two different donors. 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 4 × 10 expressing either 28 / z CAR (as measured by the frequency of cells expressing either GFP, a marker for CAR-transduced cells) 6T cells were injected intravenously. Tumor growth was assessed up to day 28 by measuring tumor volume.

[0301] Calculation of heterologous tumor growth and inhibition To measure tumor volume with external calipers, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0302] Summary of results and conclusions: A375 tumors grew progressively in mice treated with control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239 Mice receiving BB / z CAR T cells did not show any tumor control. In contrast, anti-HLA-A2 / MAGEA4 230-239 Administration of 28 / z CAR T resulted in inhibition of established A375 tumor growth at days 17 (p<0.0001 compared to control CAR T) and 20 (p<0.0001 compared to control CAR T) (statistically analyzed by two-way ANOVA). 230-239 Anti-HLA-A2 / MAGEA4 versus BB / z CAR 230-239 Enhanced efficacy of 28 / z CAR was confirmed, with tumor sizes statistically significantly different on days 13 (p=0.0256), 17 (p=0.0002), and 21 (p<0.0001) by two-way ANOVA.

[0303] [Table 27-1]

[0304] [Table 27-2]

[0305] [Table 27-3]

[0306] Incorporation by Reference All publications, patents, patent applications, and sequence reference numbers mentioned in this specification are incorporated by reference in their entireties as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including any definitions herein, will control.

[0307] Equivalent Numerous embodiments of the present invention have been described. It is understood, of course, that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.

[0308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the claims appended hereto.

Claims

1. 2. A CAR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, 9, 11, 13, 30, or 34.

2. The CAR polypeptide of claim 1 , wherein the CAR polypeptide comprises SEQ ID NO:

7.

3. 2. The CAR polypeptide of claim 1 , wherein the CAR polypeptide comprises SEQ ID NO:

9.

4. 2. The CAR polypeptide of claim 1 , wherein the CAR polypeptide comprises SEQ ID NO:

11.

5. 2. The CAR polypeptide of claim 1 , wherein the CAR polypeptide comprises SEQ ID NO:

13.

6. 2. The CAR polypeptide of claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

30.

7. 2. The CAR polypeptide of claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

34.

8. An immune cell expressing the CAR polypeptide of claim 1.

9. A composition comprising an immune cell expressing the CAR of claim 1.

10. A nucleic acid encoding the CAR polypeptide of claim 1.

11. An immune cell comprising the nucleic acid of claim 10.

12. The immune cell of claim 11, wherein the immune cell is a leukocyte, lymphocyte, monocyte, macrophage, dendritic cell, mast cell, neutrophil, basophil, eosinophil, αβT cell, γδT cell, natural killer (NK) cell, natural killer T (NKT) cell, innate lymphoid cell (ILC), cytokine-induced killer (CIK) cell, cytotoxic T lymphocyte (CTL), lymphokine-activated killer (LAK) cell, or regulatory T cell.

13. 13. A composition comprising a cell expressing the CAR polypeptide of claim 1 for use in a method of treating a tumor in a subject, the method comprising administering the composition.

14. The method comprises: a) a 4-1BB domain in the costimulatory region of the second CAR polypeptide; b) at least one cytoplasmic signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain; and c) an extracellular domain specific for an intragroove peptide cancer antigen; The composition of claim 13, further comprising co-administering to the subject the second CAR polypeptide comprising:

15. 15. The composition of claim 14, wherein the second CAR polypeptide comprises a cluster of differentiation 8 alpha (CD8 / a) peptide in its hinge / transmembrane region.

16. 2. A cell bank comprising cells for adoptive immunotherapy, wherein the cells express the CAR of claim 1.

17. 1. A nucleic acid encoding a CAR polypeptide, said nucleic acid having a sequence set forth in SEQ ID NO: 14, 15, 35, or 36.

18. 18. A nucleic acid encoding the CAR polypeptide of claim 17, wherein the nucleic acid comprises SEQ ID NO:

14.

19. 18. A nucleic acid encoding the CAR polypeptide of claim 17, wherein the nucleic acid comprises SEQ ID NO:

15.

20. 18. A nucleic acid encoding the CAR polypeptide of claim 17, wherein the nucleic acid comprises SEQ ID NO:

35.

21. 18. A nucleic acid encoding the CAR polypeptide of claim 17, wherein the nucleic acid comprises SEQ ID NO: 36.

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