CAR-T LYMPHOCYTES ENGINEERED TO HOME TO LYMPH NODE B CELL ZONE, SKIN, OR GASTROINTESTINAL TRACT
20180080008 ยท 2018-03-22
Assignee
Inventors
Cpc classification
A61K39/4632
HUMAN NECESSITIES
C12N2501/385
CHEMISTRY; METALLURGY
A61K39/4611
HUMAN NECESSITIES
C12N2501/21
CHEMISTRY; METALLURGY
C12N5/0638
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
C07K14/715
CHEMISTRY; METALLURGY
Abstract
In one embodiment, provided herein are cells, e.g., T cells expressing receptors that that cause a cell expressing the receptors to home to specific anatomical regions, e.g., the B cell zone of lymph nodes, the gastrointestinal tract, or the skin. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer.
Claims
1.-22. (canceled)
23. A method of generating a population of T lymphocytes, wherein a plurality of said T lymphocytes home to the gastrointestinal tract, said method comprising engineering a population of T lymphocytes to express a gastrointestinal homing receptor.
24. The method of claim 23, wherein the gastrointestinal homing receptor is 47.
25. The method of claim 23, wherein the gastrointestinal homing receptor is CCR9.
26. The method of claim 23, wherein the population of T lymphocytes is engineered to express a second gastrointestinal homing receptor.
27. The method of claim 23, the method further comprising a step wherein the population of T lymphocytes is activated, expanded, or both activated and expanded in the presence of a Vitamin A metabolite for a time and in an amount sufficient to cause increased expression of one or more gastrointestinal homing receptors.
28. The method of claim 27, wherein the Vitamin A metabolite is retinoic acid.
29.-42. (canceled)
43. A T lymphocyte comprising (i) a gastrointestinal homing receptor; and (ii) a chimeric antigen receptor (CAR).
44. The T lymphocyte of claim 43, wherein the gastrointestinal homing receptor is 47.
45. The T lymphocyte of claim 43, wherein the gastrointestinal homing receptor is CCR9.
46. The T lymphocyte of claim 43, wherein the T lymphocyte comprises a second gastrointestinal homing receptor.
47. The T lymphocyte claim 43, wherein the extracellular domain of the CAR binds an antigen associated with a gastrointestinal tumor or cancer.
48.-54. (canceled)
55. The T lymphocyte of claim 43, wherein the extracellular domain of the CAR binds carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, CD117, or CA 72-4.
56.-59. (canceled)
60. A method of treating a gastrointestinal cancer or tumor in an individual in need thereof, comprising administering a T lymphocyte to the individual, wherein the T lymphocyte comprises (i) a gastrointestinal homing receptor; and (ii) a CAR.
61. The method of claim 60, wherein the gastrointestinal homing receptor is 47.
62. The method of claim 60, wherein the gastrointestinal homing receptor is CCR9.
63. The method of claim 60, wherein said T lymphocyte comprises a second gastrointestinal homing receptor.
64. The method of claim 60, wherein the extracellular domain of the CAR binds an antigen associated with a gastrointestinal tumor or cancer.
65.-71. (canceled)
72. The method of claim 60, wherein the extracellular domain of the CAR binds carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, CD117, or CA 72-4.
73. (canceled)
74. (canceled)
Description
4. DETAILED DESCRIPTION
[0023] Adaptive immune responses are initiated in secondary lymphoid organs, including the lymph nodes. B cells and T cells are sequestered in distinct regions of the lymph nodes, termed the B cell zone, located in the outer cortex of the lymph node, or follicles, and the T cell zone, which is more diffusely distributed in the area surrounding the follicles (also known as the paracortex) respectively. B cells and T cells express receptors that allow them to home to these respective zones so that they can be exposed to antigen. Intact antigens are present in the B cell zone, whereas in the T cell zone, antigens are presented by antigen-presenting cells, such as dendritic cells. Intact antigens, such as tumor antigens, are also present at the site of the tumor.
[0024] Provided herein are genetically modified cells, for example immune cells, such as T lymphocytes, e.g., human T lymphocytes, that comprise a receptor that causes a cell expressing said receptor to home to a particular anatomical zone, a particular tissue, or a particular type of cell, e.g., B cell zone of the lymph nodes, gastrointestinal tract, or skin. In a specific embodiment, the genetically modified cells provided herein are T lymphocytes. In certain embodiments, the genetically modified cells express a chimeric antigen receptor (CAR), as described in Section 4.4. In certain embodiments, the genetically modified cells express a CAR and endogenously express one or more homing receptors, e.g., receptors that that cause a cell expressing the receptors to home to the B cell zone of the lymph nodes, gastrointestinal tract, or skin. Without wishing to be bound by any particular mechanism or theory, it is thought that when the genetically modified cells herein express receptors that cause a cell expressing said receptors to home to a particular zone, they are more likely to be exposed to native antigen, where the cells, for example, cells expressing a CAR, are capable of being activated.
[0025] 4.1 Cells Comprising B-Cell Zone Homing Receptors, Methods of Making, and Uses Thereof
[0026] In one embodiment, provided herein are genetically modified cells, for example immune cells, such as T lymphocytes, e.g., human T lymphocytes, that comprise, e.g., have been engineered to express, a receptor that causes the cells to home to the B cell zone of the lymph nodes, e.g., the follicles of the lymph node. Such a receptor is referred to herein as a B cell zone homing receptor. In specific embodiments, the B cell zone homing receptor is CXCR5, for example, human CXCR5. GenBank accession numbers NM_001716.4 and NM_032966.2 provide exemplary nucleotide sequences for human CXCR5. GenBank accession numbers NP_116743.1 and NP_001707.1 provide exemplary amino acid sequences for human CXCR5. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1. In a specific embodiment, the genetically modified cells provided herein are T lymphocytes. In certain embodiments, the genetically modified cells express a B cell zone homing receptor and also express a CAR, as described in Section 4.4. In certain embodiments, the genetically modified cells express a CAR and endogenously express one or more B cell zone homing receptors.
[0027] Also provided herein is a method of generating genetically engineered T lymphocytes that home to the B cell zone of a lymph node, e.g., the follicles of a lymph node, comprising a step of engineering a T lymphocyte to express a B cell zone homing receptor, e.g., CXCR5, wherein said B cell zone homing receptor is expressed by the cell at a sufficient level or sufficient amount to cause the cell to home to the B cell zone of the lymph node. In some embodiments, the step of engineering a T cell to express a B cell zone homing receptor comprises a step of introducing to the cells one or more vectors comprising the receptor nucleic acid sequence(s), i.e., the nucleic acid sequence (s) encoding the receptor(s). In specific embodiments, the vector comprises the nucleic acid sequence for human CXCR5. In a certain embodiment, the step of engineering a T cell to express a B cell zone homing receptor is performed by any method known to one of skill in the art.
[0028] Exemplary nucleic acids useful for engineering a T lymphocyte to express a B cell zone homing receptor are disclosed, for example, in Section 4.5.
[0029] Also provided herein are methods of treating a cancer or tumor in an individual comprising administering to the individual a therapeutically effective amount of genetically modified cells, e.g. human T lymphocytes, that comprise (i) a receptor that causes a cell expressing said receptor to home to a B cell zone of the lymph nodes, e.g., the follicles of the lymph node, and (ii) a CAR. In specific embodiments, the B cell zone homing receptor is CXCR5. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) a receptor that causes a cell expressing said receptor to home to a B cell zone of the lymph nodes, e.g., the follicles of the lymph node, and (ii) a CAR, suppress the proliferation of tumor cells. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) a receptor that causes a cell expressing said receptor to home to a B cell zone of the lymph nodes, e.g., the follicles of the lymph node, and (ii) a CAR, inhibit growth of the tumor. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) a receptor that causes a cell expressing said receptor to home to a B cell zone of the lymph nodes, e.g., the follicles of the lymph node, and (ii) a CAR, kill tumor cells. In specific embodiments, the extracellular domain of the CAR binds an antigen binds an antigen selected from the group consisting of Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, an abnormal p53 protein, fuc-GM1, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR fusion protein, PTPRK, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), integrin v3 (CD61), galactin, and Ral-B. Also provided herein are genetically modified cells for use in methods of treating a cancer or tumor.
[0030] 4.2. Cells Comprising Gastrointestinal Homing Receptors, Methods of Making, and Uses Thereof
[0031] In one embodiment, provided herein are genetically modified cells, for example immune cells, such as T lymphocytes, e.g., human T lymphocytes, that comprise, e.g., have been engineered to express, a receptor that causes a cell expressing said receptor to home to the gastrointestinal tract, e.g., gastrointestinal organs, tissues, or cells. Such a receptor that causes a cell to home to the gastrointestinal tract is referred to herein as a gastrointestinal homing receptor. In a specific embodiment, the genetically modified cells provided herein are T lymphocytes. In certain embodiments, the gastrointestinal homing receptor is CCR9 or integrin 47, for example, human CCR9 or human integrin 47. GenBank accession numbers NM_031200.2 and NM001256369.1 provide exemplary nucleotide sequences for human CCR9. GenBank accession numbers NP_112477.1 and NP_001243298.1 provide exemplary amino acid sequences for human CCR9. GenBank accession numbers NM_000885.4 and NM_000889.2 provide exemplary nucleotide sequences for human 4 and human 7, respectively. GenBank accession numbers NP_000876.3 and NP_000880.1 provide exemplary amino acid sequences for human 4 and human 7, respectively. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1. In some embodiments, the genetically modified cells further comprise a second gastrointestinal homing receptor. In some embodiments, the genetically modified cells comprise a first gastrointestinal homing receptor, wherein the first gastrointestinal homing receptor is CCR9, and further comprise a second gastrointestinal homing receptor, wherein the second gastrointestinal homing receptor is integrin 47. In other specific embodiments, the genetically modified cells comprise the gastrointestinal-homing receptor CXCR3.
[0032] In certain embodiments, the genetically modified cells containing one or more gastrointestinal homing receptors are expanded, activated, or both expanded and activated in the presence of a Vitamin A metabolite. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In specific embodiments, the Vitamin A metabolite is retinoic acid. In certain embodiments, the genetically modified cells containing one or more gastrointestinal homing receptors additionally comprise a B cell zone homing receptor. In specific embodiments, the B cell zone homing receptor is CXCR5. In certain embodiments, the genetically modified cells express a gastrointestinal homing receptor and also express a CAR, as described in Section 4.4. In certain embodiments, the genetically modified cells express a CAR and endogenously express one or more gastrointestinal homing receptors.
[0033] Also provided herein are methods of generating genetically modified cells, e.g. human T lymphocytes, which comprise one or more receptors that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, e.g., gastrointestinal organs, skin, or tissue. In certain embodiments, T lymphocytes comprising one or more receptors that that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, e.g., CCR9 or integrin 47, are generated by a method comprising a step of engineering a T lymphocyte to express one or more gastrointestinal homing receptors. In some embodiments, the step of engineering a T cell to express one or more gastrointestinal homing receptors comprises introducing to the cells one or more vectors comprising a nucleic acid sequence encoding the receptor. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR9, the nucleic acid sequence for human integrin 47, or both.
[0034] In certain embodiments, T lymphocytes that home to the gastrointestinal tract are generated by a method comprising a step of treating the cells with a molecule that induces the expression of one or more gastrointestinal homing receptors, e.g., CCR9 or 47. In specific embodiments, the molecule is Vitamin A.
[0035] In certain embodiments, the method for generating the genetically modified T lymphocytes that comprise one or more receptors that that cause a cell expressing the one or more receptors to home to the gastrointestinal tract comprises a step of expanding the cells, which step is carried out in the presence of a vitamin A metabolite. In certain embodiments, the method for generating the genetically modified T lymphocytes that comprise one or more receptors homing to the gastrointestinal tract comprises a step of activating the cells, which step is carried out in the presence of a vitamin A metabolite. In certain embodiments, both the expanding and activating steps are carried out in the presence of a vitamin A metabolite. In certain embodiments the vitamin A metabolite is retinoic acid. In a certain embodiment, the step of engineering a T cell to express a gastrointestinal homing receptor is performed by any method known to one of skill in the art.
[0036] Exemplary nucleic acids useful for engineering a T lymphocyte to express a gastrointestinal homing receptor are discussed, for example, in Section 4.5.
[0037] Also provided herein are methods of treating a gastrointestinal cancer or tumor in an individual comprising administering to the individual a therapeutically effective amount of genetically modified cells, e.g. human T lymphocytes that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, and (ii) a CAR. In certain embodiments, the gastrointestinal homing receptor is CCR9 or 47. In some embodiments, the genetically modified cells further comprise a second gastrointestinal homing receptor. In some embodiments, the genetically modified cells comprise a first gastrointestinal homing receptor, wherein the first gastrointestinal homing receptor is CCR9, and further comprise a second gastrointestinal homing receptor, wherein the second gastrointestinal homing receptor is 47. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, and (ii) a CAR, suppress the proliferation of tumor cells. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, and (ii) a CAR, inhibit growth of the tumor. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, and (ii) a CAR, kill tumor cells. In specific embodiments, the gastrointestinal cancer or tumor is liver cancer, stomach cancer, esophageal cancer, gallbladder cancer, colorectal cancer, anal cancer, or pancreatic cancer. In certain embodiments, the extracellular domain of the CAR binds an antigen associated with a gastrointestinal cancer or tumor. Also provided herein are genetically modified cells for use in methods of treating a gastrointestinal cancer or tumor.
[0038] 4.3. Cells Comprising Skin Homing Receptors, Methods of Making, and Uses Thereof
[0039] In one embodiment, provided herein are genetically modified cells, for example immune cells, such as T lymphocytes, e.g., human T lymphocytes, that comprise a receptor that causes a cell expressing said receptor to home to the skin, e.g., skin tissue, or skin cells. In a specific embodiment, the genetically modified cells provided herein are T lymphocytes. In certain embodiments, the skin homing receptor is CCR10, CCR8, CCR4, or CLA, for example, human CCR10, human CCR8, human CCR4, or human CLA. GenBank accession numbers NM_016602.2 and AF215981.1 provide exemplary nucleotide sequences for human CCR10. GenBank accession numbers NP_057686.2 and P46092.3 provide exemplary amino acid sequences for human CCR10. GenBank accession numbers NM_005201.3 and BC107159.1 provide exemplary nucleotide sequences for human CCR8. GenBank accession numbers NP_005192.1 and AAI07160.1 provide exemplary amino acid sequences for human CCR8. GenBank accession number NM_005508.4 provides an exemplary nucleotide sequence for human CCR4. GenBank accession number P51679.1 provides an exemplary amino acid sequence for human CCR4. GenBank accession numbers NM_001206609.1 and NM_003006.4 provide exemplary nucleotide sequences for human CLA. GenBank accession numbers NP_001193538.1 and NP_002997.2 provide exemplary amino acid sequences for human CLA. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1. In some embodiments, the genetically modified cells further comprise a second skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR4, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the genetically modified cells further comprise a third skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR8, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA, CCR4, or CCR10. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR8, further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA, CCR4, or CCR10, and further comprise a third skin homing receptor, wherein the third skin homing receptor is distinct from the second skin homing receptor, and is selected from the group consisting of CLA, CCR4, and CCR10. In some embodiments, the genetically modified cells further comprise a third skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA, and further comprise a fourth skin homing receptor, wherein the fourth skin homing receptor is CCR8. In certain embodiments, the genetically modified cells comprise one or more skin homing receptors. In other specific embodiments, the genetically modified cells comprise the skin-homing receptor CCR6.
[0040] In certain embodiments, the genetically modified cells containing one or more skin homing receptors are expanded, activated, or both expanded and activated in the presence of a Vitamin D metabolite. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In specific embodiments, the Vitamin D metabolite is 1,25-dihydroxycholecalciferol (1,25(OH).sub.2D.sub.3). In certain embodiments, the genetically modified cells containing one or more skin homing receptors are expanded, activated, or both expanded and activated in the presence of IL-12. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In more specific embodiments, the genetically modified cells containing one or more skin homing receptors are expanded, activated, or both expanded and activated in the presence of a Vitamin D metabolite and IL-12. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In certain embodiments, the genetically modified cells containing one or more skin homing receptors additionally comprise a B cell zone homing receptor. In specific embodiments, the B cell zone homing receptor is CXCR5. In certain embodiments, the genetically modified cells express a skin homing receptor and also express a CAR, as described in Section 4.4. In certain embodiments, the genetically modified cells express a CAR and endogenously express one or more skin homing receptors.
[0041] Also provided herein are methods of generating genetically modified cells, e.g. human T lymphocytes, that comprise one or more receptors homing to the skin, e.g., skin tissue or cells. In certain embodiments, T lymphocytes that home to the skin are generated by a method comprising a step of engineering the T lymphocytes to express a skin homing receptor, e.g., CCR4, CCR8, CCR10, or CLA. In some embodiments, the step of engineering the T lymphocytes to express a skin homing receptor comprises introducing into the cells one or more vectors comprising the receptor nucleic acid sequence(s), i.e., the nucleic acid sequence(s) encoding the receptor(s). In specific embodiments, the vector comprises the nucleic acid sequence for human CCR10, the nucleic acid sequence for human CLA, or both. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR4, and optionally the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR4 and the nucleic acid sequence for human CCR10. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR10, the nucleic acid sequence for human CCR4, and the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, and optionally the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8 and the nucleic acid sequence for human CCR10. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR4, and the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR10, and the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR4, and the nucleic acid sequence for human CCR10. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR4, the nucleic acid for CCR10, and the nucleic acid sequence for human CLA.
[0042] In certain embodiments, cells, e.g., T lymphocytes, that home to the skin are generated by a method comprising a step of treating the cells, e.g., T lymphocytes, with a molecule that induces, e.g., increases, the expression of one or more skin homing receptors, e.g., CCR4, CCR10, CCR8, or CLA. In specific embodiments, the molecule is Vitamin D. In certain embodiments, the induction of expression of skin homing receptors is aided by treating the cells, e.g., T lymphocytes, with IL-12, e.g., contacting the cells with IL-12 in an amount and for a time sufficient to increase expression of one or more of CCR4, CCR8, CCR10, or CLA by said cells.
[0043] In certain embodiments, the method for generating the genetically modified T lymphocytes that comprise one or more receptors that cause a cell expressing the one or more receptors to home to the skin, comprises a step of expanding the cells, which step is carried out in the presence of a vitamin D metabolite and, optionally, IL-12. In certain embodiments, the method for generating the genetically modified T lymphocytes that comprise one or more receptors that that cause a cell expressing the one or more receptors to home to the gastrointestinal tract, comprises a step of activating the cells, which step is carried out in the presence of a vitamin D metabolite, and, optionally, IL-12. In certain embodiments, both the expanding and activating steps are carried out in the presence of a vitamin D metabolite, and, optionally, IL-12. In certain embodiments the vitamin D metabolite is 1,25(OH).sub.2D.sub.3. In a certain embodiment, the step of engineering a T cell to express a skin homing receptor is performed by any method known to one of skill in the art.
[0044] Exemplary nucleic acids useful for engineering a T lymphocyte to express a skin homing receptor are discussed, for example, in Section 4.5.
[0045] Also provided herein are methods of treating a skin cancer or tumor in an individual comprising administering to the individual a therapeutically effective amount of genetically modified cells, e.g. human T lymphocytes that comprise (i) one or more receptors that that cause a cell expressing the one or more receptors to home to the skin, and (ii) a CAR. In certain embodiments, the skin homing receptor is CCR10, CCR8, CCR4, or CLA. In some embodiments, the genetically modified cells further comprise a second skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR8. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR4, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR4, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR8. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CLA, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR8. In some embodiments, the genetically modified cells further comprise a third skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CCR8. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR8, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR8, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA. In some embodiments, the genetically modified cells further comprise a fourth skin homing receptor. In some embodiments, the genetically modified cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR10, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA, and further comprise a fourth skin homing receptor, wherein the fourth skin homing receptor is CCR8. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the skin, and (ii) a CAR, suppress the proliferation of tumor cells. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the skin, and (ii) a CAR, inhibit growth of the tumor. In certain embodiments, the genetically modified cells, e.g. human T lymphocytes, that comprise (i) one or more receptors that cause a cell expressing the one or more receptors to home to the skin, and (ii) a CAR, kill tumor cells. In specific embodiments, the skin cancer or tumor is melanoma, squamous cell carcinoma, or basal cell carcinoma. In certain embodiments, the extracellular domain of the CAR binds an antigen associated with a skin cancer or tumor. Also provided herein are genetically modified cells for use in methods of treating a skin cancer or tumor.
TABLE-US-00001 TABLE1 Exemplarynucleotideandaminoacidsequencesforhumanhomingreceptors. GenBank SEQ Accession ID Numberand NO: Description Sequence 1 NM_001716.4 1aaaaaaaaaaagtgatgagttgtgaggcaggtcgcggccctactgcctcaggagacgatg Exemplarynucleic 61cgcagctcatttgcttaaatttgcagctgacggctgccacctctctagaggcacctggcg acidsequence 121gggagcctctcaacataagacagtgaccagtctggtgactcacagccggcacagccatga encodinghuman 181actacccgctaacgctggaaatggacctcgagaacctggaggacctgttctgggaactgg CXCR5 241acagattggacaactataacgacacctccctggtggaaaatcatctctgccctgccacag 301aggggcccctcatggcctccttcaaggccgtgttcgtgcccgtggcctacagcctcatct 361tcctcctgggcgtgatcggcaacgtcctggtgctggtgatcctggagcggcaccggcaga 421cacgcagttccacggagaccttcctgttccacctggccgtggccgacctcctgctggtct 481tcatcttgccctttgccgtggccgagggctctgtgggctgggtcctggggaccttcctct 541gcaaaactgtgattgccctgcacaaagtcaacttctactgcagcagcctgctcctggcct 601gcatcgccgtggaccgctacctggccattgtccacgccgtccatgcctaccgccaccgcc 661gcctcctctccatccacatcacctgtgggaccatctggctggtgggcttcctccttgcct 721tgccagagattctcttcgccaaagtcagccaaggccatcacaacaactccctgccacgtt 781gcaccttctcccaagagaaccaagcagaaacgcatgcctggttcacctcccgattcctct 841accatgtggcgggattcctgctgcccatgctggtgatgggctggtgctacgtgggggtag 901tgcacaggttgcgccaggcccagcggcgccctcagcggcagaaggcagtcagggtggcca 961tcctggtgacaagcatcttcttcctctgctggtcaccctaccacatcgtcatcttcctgg 1021acaccctggcgaggctgaaggccgtggacaatacctgcaagctgaatggctctctccccg 1081tggccatcaccatgtgtgagttcctgggcctggcccactgctgcctcaaccccatgctct 1141acactttcgccggcgtgaagttccgcagtgacctgtcgcggctcctgacgaagctgggct 1201gtaccggccctgcctccctgtgccagctcttccctagctggcgcaggagcagtctctctg 1261agtcagagaatgccacctctctcaccacgttctaggtcccagtgtccccttttattgctg 1321cttttccttggggcaggcagtgatgctggatgctccttccaacaggagctgggatcctaa 1381gggctcaccgtggctaagagtgtcctaggagtatcctcatttggggtagctagaggaacc 1441aacccccatttctagaacatccctgccagctcttctgccggccctggggctaggctggag 1501cccagggagcggaaagcagctcaaaggcacagtgaaggctgtccttacccatctgcaccc 1561ccctgggctgagagaacctcacgcacctcccatcctaatcatccaatgctcaagaaacaa 1621cttctacttctgcccttgccaacggagagcgcctgcccctcccagaacacactccatcag 1681cttaggggctgctgacctccacagcttcccctctctcctcctgcccacctgtcaaacaaa 1741gccagaagctgagcaccaggggatgagtggaggttaaggctgaggaaaggccagctggca 1801gcagagtgtggccttcggacaactcagtccctaaaaacacagacattctgccaggccccc 1861aagcctgcagtcatcttgaccaagcaggaagctcagactggttgagttcaggtagctgcc 1921cctggctctgaccgaaacagcgctgggtccaccccatgtcaccggatcctgggtggtctg 1981caggcagggctgactctaggtgcccttggaggccagccagtgacctgaggaagcgtgaag 2041gccgagaagcaagaaagaaacccgacagagggaagaaaagagctttcttcccgaacccca 2101aggagggagatggatcaatcaaacccggcggtcccctccgccaggcgagatggggtgggg 2161tggagaactcctagggtggctgggtccaggggatgggaggttgtgggcattgatggggaa 2221ggaggctggcttgtcccctcctcactcccttcccataagctatagacccgaggaaactca 2281gagtcggaacggagaaaggtggactggaaggggcccgtgggagtcatctcaaccatcccc 2341tccgtggcatcaccttaggcagggaagtgtaagaaacacactgaggcagggaagtcccca 2401ggccccaggaagccgtgccctgcccccgtgaggatgtcactcagatggaaccgcaggaag 2461ctgctccgtgcttgtttgctcacctggggtgtgggaggcccgtccggcagttctgggtgc 2521tccctaccacctccccagcctttgatcaggtggggagtcagggacccctgcccttgtccc 2581actcaagccaagcagccaagctccttgggaggccccactggggaaataacagctgtggct 2641cacgtgagagtgtcttcacggcaggacaacgaggaagccctaagacgtcccttttttctc 2701tgagtatctcctcgcaagctgggtaatcgatgggggagtctgaagcagatgcaaagaggc 2761aagaggctggattttgaattttctttttaataaaaaggcacctataaaacaggtcaatac 2821agtacaggcagcacagagacccccggaacaagcctaaaaattgtttcaaaataaaaacca 2881agaagatgtcttcacatattgtaaaaaaaaaaaaaaaaa 2 NM_032966.2 1ccactctaaggaatgcggtccctttgacaggcgaaaaactgaagttggaaaagacaaagt Exemplarynucleic 61gatttgttcaaaattgaaatttgaaacttgacatttggtcagtgggccctatgtaggaaa acidsequence 121aaacctccaagagagctagggttcctctcagagaggaaagacaggtccttaggtcctcac encodinghuman 181cctcccgtctccttgcccttgcagttctgggaactggacagattggacaactataacgac CXCR5 241acctccctggtggaaaatcatctctgccctgccacagaggggcccctcatggcctccttc 301aaggccgtgttcgtgcccgtggcctacagcctcatcttcctcctgggcgtgatcggcaac 361gtcctggtgctggtgatcctggagcggcaccggcagacacgcagttccacggagaccttc 421ctgttccacctggccgtggccgacctcctgctggtcttcatcttgccctttgccgtggcc 481gagggctctgtgggctgggtcctggggaccttcctctgcaaaactgtgattgccctgcac 541aaagtcaacttctactgcagcagcctgctcctggcctgcatcgccgtggaccgctacctg 601gccattgtccacgccgtccatgcctaccgccaccgccgcctcctctccatccacatcacc 661tgtgggaccatctggctggtgggcttcctccttgccttgccagagattctcttcgccaaa 721gtcagccaaggccatcacaacaactccctgccacgttgcaccttctcccaagagaaccaa 781gcagaaacgcatgcctggttcacctcccgattcctctaccatgtggcgggattcctgctg 841cccatgctggtgatgggctggtgctacgtgggggtagtgcacaggttgcgccaggcccag 901cggcgccctcagcggcagaaggcagtcagggtggccatcctggtgacaagcatcttcttc 961ctctgctggtcaccctaccacatcgtcatcttcctggacaccctggcgaggctgaaggcc 1021gtggacaatacctgcaagctgaatggctctctccccgtggccatcaccatgtgtgagttc 1081ctgggcctggcccactgctgcctcaaccccatgctctacactttcgccggcgtgaagttc 1141cgcagtgacctgtcgcggctcctgacgaagctgggctgtaccggccctgcctccctgtgc 1201cagctcttccctagctggcgcaggagcagtctctctgagtcagagaatgccacctctctc 1261accacgttctaggtcccagtgtccccttttattgctgcttttccttggggcaggcagtga 1321tgctggatgctccttccaacaggagctgggatcctaagggctcaccgtggctaagagtgt 1381cctaggagtatcctcatttggggtagctagaggaaccaacccccatttctagaacatccc 1441tgccagctcttctgccggccctggggctaggctggagcccagggagcggaaagcagctca 1501aaggcacagtgaaggctgtccttacccatctgcacccccctgggctgagagaacctcacg 1561cacctcccatcctaatcatccaatgctcaagaaacaacttctacttctgcccttgccaac 1621ggagagcgcctgcccctcccagaacacactccatcagcttaggggctgctgacctccaca 1681gcttcccctctctcctcctgcccacctgtcaaacaaagccagaagctgagcaccagggga 1741tgagtggaggttaaggctgaggaaaggccagctggcagcagagtgtggccttcggacaac 1801tcagtccctaaaaacacagacattctgccaggcccccaagcctgcagtcatcttgaccaa 1861gcaggaagctcagactggttgagttcaggtagctgcccctggctctgaccgaaacagcgc 1921tgggtccaccccatgtcaccggatcctgggtggtctgcaggcagggctgactctaggtgc 1981ccttggaggccagccagtgacctgaggaagcgtgaaggccgagaagcaagaaagaaaccc 2041gacagagggaagaaaagagctttcttcccgaaccccaaggagggagatggatcaatcaaa 2101cccggcggtcccctccgccaggcgagatggggtggggtggagaactcctagggtggctgg 2161gtccaggggatgggaggttgtgggcattgatggggaaggaggctggcttgtcccctcctc 2221actcccttcccataagctatagacccgaggaaactcagagtcggaacggagaaaggtgga 2281ctggaaggggcccgtgggagtcatctcaaccatcccctccgtggcatcaccttaggcagg 2341gaagtgtaagaaacacactgaggcagggaagtccccaggccccaggaagccgtgccctgc 2401ccccgtgaggatgtcactcagatggaaccgcaggaagctgctccgtgcttgtttgctcac 2461ctggggtgtgggaggcccgtccggcagttctgggtgctccctaccacctccccagccttt 2521gatcaggtggggagtcagggacccctgcccttgtcccactcaagccaagcagccaagctc 2581cttgggaggccccactggggaaataacagctgtggctcacgtgagagtgtcttcacggca 2641ggacaacgaggaagccctaagacgtcccttttttctctgagtatctcctcgcaagctggg 2701taatcgatgggggagtctgaagcagatgcaaagaggcaagaggctggattttgaattttc 2761tttttaataaaaaggcacctataaaacaggtcaatacagtacaggcagcacagagacccc 2821cggaacaagcctaaaaattgtttcaaaataaaaaccaagaagatgtcttcacatattgta 2881aaaaaaaaaaaaaaaa 3 NP_116743.1 1masfkavfvpvayslifllgvignvlvlvilerhrqtrsstetflfhlavadlllvfilp Exemplaryamino 61favaegsvgwvlgtflcktvialhkvnfycsslllaciavdrylaivhavhayrhrrlls acidsequencefor 121ihitcgtiwlvgfllalpeilfakvsqghhnnslprctfsqenqaethawftsrflyhva humanCXCR5 181gfllpmlvmgwcyvgvvhrlrqaqrrpqrqkavrvailvtsifflcwspyhivifldtla precursor 241rlkavdntcklngslpvaitmceflglahcclnpmlytfagvkfrsdlsrlltklgctgp 301aslcqlfpswrrsslsesenatslttf 4 NP_001707.1 1mnypltlemdlenledlfweldrldnyndtslvenhlcpategplmasfkavfvpvaysl Exemplaryamino 61ifllgvignvlvlvilerhrqtrsstetflfhlavadlllvfilpfavaegsvgwvlgtf acidsequencefor 121lcktvialhkvnfycsslllaciavdrylaivhavhayrhrrllsihitcgtiwlvgfll humanCXCR5 181alpeilfakvsqghhnnslprctfsqenqaethawftsrflyhvagfllpmlvmgwcyvg precursor 241vvhrlrqaqrrpqrqkavrvailvtsifflcwspyhivifldtlarlkavdntcklngsl 301pvaitmceflglahcclnpmlytfagvkfrsdlsrlltklgctgpaslcqlfpswrrssl 361sesenatslttf 5 NM_031200.2 1gcttcctttctcgtgttgttatcgggtagctgcctgctcagaacccacaaagcctgcccc Exemplarynucleic 61tcatcccaggcagagagcaacccagctctttccccagacactgagagctggtggtgcctg acidsequence 121ctgtcccagggagagttgcatcgccctccacagagcaggcttgcatctgactgacccacc encodinghuman 181atgacacccacagacttcacaagccctattcctaacatggctgatgactatggctctgaa CCR9 241tccacatcttccatggaagactacgttaacttcaacttcactgacttctactgtgagaaa 301aacaatgtcaggcagtttgcgagccatttcctcccacccttgtactggctcgtgttcatc 361gtgggtgccttgggcaacagtcttgttatccttgtctactggtactgcacaagagtgaag 421accatgaccgacatgttccttttgaatttggcaattgctgacctcctctttcttgtcact 481cttcccttctgggccattgctgctgctgaccagtggaagttccagaccttcatgtgcaag 541gtggtcaacagcatgtacaagatgaacttctacagctgtgtgttgctgatcatgtgcatc 601agcgtggacaggtacattgccattgcccaggccatgagagcacatacttggagggagaaa 661aggcttttgtacagcaaaatggtttgctttaccatctgggtattggcagctgctctctgc 721atcccagaaatcttatacagccaaatcaaggaggaatccggcattgctatctgcaccatg 781gtttaccctagcgatgagagcaccaaactgaagtcagctgtcttgaccctgaaggtcatt 841ctggggttcttccttcccttcgtggtcatggcttgctgctataccatcatcattcacacc 901ctgatacaagccaagaagtcttccaagcacaaagccctaaaagtgaccatcactgtcctg 961accgtctttgtcttgtctcagtttccctacaactgcattttgttggtgcagaccattgac 1021gcctatgccatgttcatctccaactgtgccgtttccaccaacattgacatctgcttccag 1081gtcacccagaccatcgccttcttccacagttgcctgaaccctgttctctatgtttttgtg 1141ggtgagagattccgccgggatctcgtgaaaaccctgaagaacttgggttgcatcagccag 1201gcccagtgggtttcatttacaaggagagagggaagcttgaagctgtcgtctatgttgctg 1261gagacaacctcaggagcactctccctctgaggggtcttctctgaggtgcatggttctttt 1321ggaagaaatgagaaatacagaaacagtttccccactgatgggaccagagagagtgaaaga 1381gaaaagaaaactcagaaagggatgaatctgaactatatgattacttgtagtcagaatttg 1441ccaaagcaaatatttcaaaatcaactgactagtgcaggaggctgttgattggctcttgac 1501tgtgatgcccgcaattctcaaaggaggactaaggaccggcactgtggagcaccctggctt 1561tgccactcgccggagcatcaatgccgctgcctctggaggagcccttggattttctccatg 1621cactgtgaacttctgtggcttcagttctcatgctgcctcttccaaaaggggacacagaag 1681cactggctgctgctacagaccgcaaaagcagaaagtttcgtgaaaatgtccatctttggg 1741aaattttctaccctgctcttgagcctgataacccatgccaggtcttatagattcctgatc 1801tagaacctttccaggcaatctcagacctaatttccttctgttctccttgttctgttctgg 1861gccagtgaaggtccttgttctgattttgaaacgatctgcaggtcttgccagtgaacccct 1921ggacaactgaccacacccacaaggcatccaaagtctgttggcttccaatccatttctgtg 1981tcctgctggaggttttaacctagacaaggattccgcttattccttggtatggtgacagtg 2041tctctccatggcctgagcagggagattataacagctgggttcgcaggagccagccttggc 2101cctgttgtaggcttgttctgttgagtggcacttgctttgggtccaccgtctgtctgctcc 2161ctagaaaatgggctggttcttttggccctcttctttctgaggcccactttattctgagga 2221atacagtgagcagatatgggcagcagccaggtagggcaaaggggtgaagcgcaggccttg 2281ctggaaggctatttacttccatgcttctccttttcttactctatagtggcaacattttaa 2341aagcttttaacttagagattaggctgaaaaaaataagtaatggaattcacctttgcatct 2401tttgtgtctttcttatcatgatttggcaaaatgcatcacctttgaaaatatttcacatat 2461tggaaaagtgctttttaatgtgtatatgaagcattaattacttgtcactttctttaccct 2521gtctcaatattttaagtgtgtgcaattaaagatcaaatagatacatt 6 NM001256369.1 1gcttcctttctcgtgttgttatcgggtagctgcctgctcagaacccacaaagcctgcccc Exemplarynucleic 61tcatcccaggcagagagcaacccagctctttccccagacactgagagctggtggtgcctg acidsequence 121ctgtcccagggagagttgcatcgccctccacagagcaggcttgcatctgactgacccacc encodinghuman 181atgacacccacagacttcacatctcctccaggccccgctccagatcaccttccctcgctg CCR9 241gcccaggaatccatctccttccaggaccttagcccaggactaacacaagccctattccta 301acatggctgatgactatggctctgaatccacatcttccatggaagactacgttaacttca 361acttcactgacttctactgtgagaaaaacaatgtcaggcagtttgcgagccatttcctcc 421cacccttgtactggctcgtgttcatcgtgggtgccttgggcaacagtcttgttatccttg 481tctactggtactgcacaagagtgaagaccatgaccgacatgttccttttgaatttggcaa 541ttgctgacctcctctttcttgtcactcttcccttctgggccattgctgctgctgaccagt 601ggaagttccagaccttcatgtgcaaggtggtcaacagcatgtacaagatgaacttctaca 661gctgtgtgttgctgatcatgtgcatcagcgtggacaggtacattgccattgcccaggcca 721tgagagcacatacttggagggagaaaaggcttttgtacagcaaaatggtttgctttacca 781tctgggtattggcagctgctctctgcatcccagaaatcttatacagccaaatcaaggagg 841aatccggcattgctatctgcaccatggtttaccctagcgatgagagcaccaaactgaagt 901cagctgtcttgaccctgaaggtcattctggggttcttccttcccttcgtggtcatggctt 961gctgctataccatcatcattcacaccctgatacaagccaagaagtcttccaagcacaaag 1021ccctaaaagtgaccatcactgtcctgaccgtctttgtcttgtctcagtttccctacaact 1081gcattttgttggtgcagaccattgacgcctatgccatgttcatctccaactgtgccgttt 1141ccaccaacattgacatctgcttccaggtcacccagaccatcgccttcttccacagttgcc 1201tgaaccctgttctctatgtttttgtgggtgagagattccgccgggatctcgtgaaaaccc 1261tgaagaacttgggttgcatcagccaggcccagtgggtttcatttacaaggagagagggaa 1321gcttgaagctgtcgtctatgttgctggagacaacctcaggagcactctccctctgagggg 1381tcttctctgaggtgcatggttcttttggaagaaatgagaaatacagaaacagtttcccca 1441ctgatgggaccagagagagtgaaagagaaaagaaaactcagaaagggatgaatctgaact 1501atatgattacttgtagtcagaatttgccaaagcaaatatttcaaaatcaactgactagtg 1561caggaggctgttgattggctcttgactgtgatgcccgcaattctcaaaggaggactaagg 1621accggcactgtggagcaccctggctttgccactcgccggagcatcaatgccgctgcctct 1681ggaggagcccttggattttctccatgcactgtgaacttctgtggcttcagttctcatgct 1741gcctcttccaaaaggggacacagaagcactggctgctgctacagaccgcaaaagcagaaa 1801gtttcgtgaaaatgtccatctttgggaaattttctaccctgctcttgagcctgataaccc 1861atgccaggtcttatagattcctgatctagaacctttccaggcaatctcagacctaatttc 1921cttctgttctccttgttctgttctgggccagtgaaggtccttgttctgattttgaaacga 1981tctgcaggtcttgccagtgaacccctggacaactgaccacacccacaaggcatccaaagt 2041ctgttggcttccaatccatttctgtgtcctgctggaggttttaacctagacaaggattcc 2101gcttattccttggtatggtgacagtgtctctccatggcctgagcagggagattataacag 2161ctgggttcgcaggagccagccttggccctgttgtaggcttgttctgttgagtggcacttg 2221ctttgggtccaccgtctgtctgctccctagaaaatgggctggttcttttggccctcttct 2281ttctgaggcccactttattctgaggaatacagtgagcagatatgggcagcagccaggtag 2341ggcaaaggggtgaagcgcaggccttgctggaaggctatttacttccatgcttctcctttt 2401cttactctatagtggcaacattttaaaagcttttaacttagagattaggctgaaaaaaat 2461aagtaatggaattcacctttgcatcttttgtgtctttcttatcatgatttggcaaaatgc 2521atcacctttgaaaatatttcacatattggaaaagtgctttttaatgtgtatatgaagcat 2581taattacttgtcactttctttaccctgtctcaatattttaagtgtgtgcaattaaagatc 2641aaatagatacatt 7 NP_112477.1 1mtptdftspipnmaddygsestssmedyvnfnftdfyceknnvrqfashflpplywlvfi Exemplaryamino 61vgalgnslvilvywyctrvktmtdmfllnlaiadllflvtlpfwaiaaadqwkfqtfmck acidsequencefor 121vvnsmykmnfyscvllimcisvdryiaiaqamrahtwrekrllyskmvcftiwvlaaalc humanCCR9 181ipeilysqikeesgiaictmvypsdestklksavltlkvilgfflpfvvmaccytiiiht precursor 241liqakksskhkalkvtitvltvfvlsqfpyncillvqtidayamfisncavstnidicfq 301vtqtiaffhsclnpvlyvfvgerfrrdlvktlknlgcisqaqwvsftrregslklssmll 361ettsgalsl 8 NP_001243298.1 1maddygsestssmedyvnfnftdfyceknnvrqfashflpplywlvfivgalgnslvilv Exemplaryamino 61ywyctrvktmtdmfllnlaiadllflvtlpfwaiaaadqwkfqtfmckvvnsmykmnfys acidsequencefor 121cvllimcisvdryiaiaqamrahtwrekrllyskmvcftiwvlaaalcipeilysqikee humanCCR9 181sgiaictmvypsdestklksavltlkvilgfflpfvvmaccytiiihtliqakksskhka precursor 241lkvtitvltvfvlsqfpyncillvqtidayamfisncavstnidicfqvtqtiaffhscl 301npvlyvfvgerfrrdlvktlknlgcisqaqwvsftrregslklssmllettsgalsl 9 NM_000885.4 1ataacgtctttgtcactaaaatgttccccaggggccttcggcgagtctttttgtttggtt Exemplarynucleic 61ttttgtttttaatctgtggctcttgataatttatctagtggttgcctacacctgaaaaac acidsequence 121aagacacagtgtttaactatcaacgaaagaactggacggctccccgccgcagtcccactc encodinghuman 181cccgagtttgtggctggcatttgggccacgccgggctgggcggtcacagcgaggggcgcg 4 241cagtttggggtcacacagctccgcttctaggccccaaccaccgttaaaaggggaagcccg 301tgccccatcaggtccgctcttgctgagcccagagccatcccgcgctctgcgggctgggag 361gcccgggccaggacgcgagtcctgcgcagccgaggttccccagcgccccctgcagccgcg 421cgtaggcagagacggagcccggccctgcgcctccgcaccacgcccgggaccccacccagc 481ggcccgtacccggagaagcagcgcgagcacccgaagctcccggctggcggcagaaaccgg 541gagtggggccgggcgagtgcgcggcatcccaggccggcccgaacgctccgcccgcggtgg 601gccgacttcccctcctcttccctctctccttcctttagcccgctggcgccggacacgctg 661cgcctcatctcttggggcgttcttccccgttggccaaccgtcgcatcccgtgcaactttg 721gggtagtggccgtttagtgttgaatgttccccaccgagagcgcatggcttgggaagcgag 781gcgcgaacccggcccccgaagggccgccgtccgggagacggtgatgctgttgctgtgcct 841gggggtcccgaccggccgcccctacaacgtggacactgagagcgcgctgctttaccaggg 901cccccacaacacgctgttcggctactcggtcgtgctgcacagccacggggcgaaccgatg 961gctcctagtgggtgcgcccactgccaactggctcgccaacgcttcagtgatcaatcccgg 1021ggcgatttacagatgcaggatcggaaagaatcccggccagacgtgcgaacagctccagct 1081gggtagccctaatggagaaccttgtggaaagacttgtttggaagagagagacaatcagtg 1141gttgggggtcacactttccagacagccaggagaaaatggatccatcgtgacttgtgggca 1201tagatggaaaaatatattttacataaagaatgaaaataagctccccactggtggttgcta 1261tggagtgccccctgatttacgaacagaactgagtaaaagaatagctccgtgttatcaaga 1321ttatgtgaaaaaatttggagaaaattttgcatcatgtcaagctggaatatccagttttta 1381cacaaaggatttaattgtgatgggggccccaggatcatcttactggactggctctctttt 1441tgtctacaatataactacaaataaatacaaggcttttttagacaaacaaaatcaagtaaa 1501atttggaagttatttaggatattcagtcggagctggtcattttcggagccagcatactac 1561cgaagtagtcggaggagctcctcaacatgagcagattggtaaggcatatatattcagcat 1621tgatgaaaaagaactaaatatcttacatgaaatgaaaggtaaaaagcttggatcgtactt 1681tggagcttctgtctgtgctgtggacctcaatgcagatggcttctcagatctgctcgtggg 1741agcacccatgcagagcaccatcagagaggaaggaagagtgtttgtgtacatcaactctgg 1801ctcgggagcagtaatgaatgcaatggaaacaaacctcgttggaagtgacaaatatgctgc 1861aagatttggggaatctatagttaatcttggcgacattgacaatgatggctttgaagatgt 1921tgctatcggagctccacaagaagatgacttgcaaggtgctatttatatttacaatggccg 1981tgcagatgggatctcgtcaaccttctcacagagaattgaaggacttcagatcagcaaatc 2041gttaagtatgtttggacagtctatatcaggacaaattgatgcagataataatggctatgt 2101agatgtagcagttggtgcttttcggtctgattctgctgtcttgctaaggacaagacctgt 2161agtaattgttgacgcttctttaagccaccctgagtcagtaaatagaacgaaatttgactg 2221tgttgaaaatggatggccttctgtgtgcatagatctaacactttgtttctcatataaggg 2281caaggaagttccaggttacattgttttgttttataacatgagtttggatgtgaacagaaa 2341ggcagagtctccaccaagattctatttctcttctaatggaacttctgacgtgattacagg 2401aagcatacaggtgtccagcagagaagctaactgtagaacacatcaagcatttatgcggaa 2461agatgtgcgggacatcctcaccccaattcagattgaagctgcttaccaccttggtcctca 2521tgtcatcagtaaacgaagtacagaggaattcccaccacttcagccaattcttcagcagaa 2581gaaagaaaaagacataatgaaaaaaacaataaactttgcaaggttttgtgcccatgaaaa 2641ttgttctgctgatttacaggtttctgcaaagattgggtttttgaagccccatgaaaataa 2701aacatatcttgctgttgggagtatgaagacattgatgttgaatgtgtccttgtttaatgc 2761tggagatgatgcatatgaaacgactctacatgtcaaactacccgtgggtctttatttcat 2821taagattttagagctggaagagaagcaaataaactgtgaagtcacagataactctggcgt 2881ggtacaacttgactgcagtattggctatatatatgtagatcatctctcaaggatagatat 2941tagctttctcctggatgtgagctcactcagcagagcggaagaggacctcagtatcacagt 3001gcatgctacctgtgaaaatgaagaggaaatggacaatctaaagcacagcagagtgactgt 3061agcaatacctttaaaatatgaggttaagctgactgttcatgggtttgtaaacccaacttc 3121atttgtgtatggatcaaatgatgaaaatgagcctgaaacgtgcatggtggagaaaatgaa 3181cttaactttccatgttatcaacactggcaatagtatggctcccaatgttagtgtggaaat 3241aatggtaccaaattcttttagcccccaaactgataagctgttcaacattttggatgtcca 3301gactactactggagaatgccactttgaaaattatcaaagagtgtgtgcattagagcagca 3361aaagagtgcaatgcagaccttgaaaggcatagtccggttcttgtccaagactgataagag 3421gctattgtactgcataaaagctgatccacattgtttaaatttcttgtgtaattttgggaa 3481aatggaaagtggaaaagaagccagtgttcatatccaactggaaggccggccatccatttt 3541agaaatggatgagacttcagcactcaagtttgaaataagagcaacaggttttccagagcc 3601aaatccaagagtaattgaactaaacaaggatgagaatgttgcgcatgttctactggaagg 3661actacatcatcaaagacccaaacgttatttcaccatagtgattatttcaagtagcttgct 3721acttggacttattgtacttctgttgatctcatatgttatgtggaaggctggcttctttaa 3781aagacaatacaaatctatcctacaagaagaaaacagaagagacagttggagttatatcaa 3841cagtaaaagcaatgatgattaaggacttctttcaaattgagagaatggaaaacagactca 3901ggttgtagtaaagaaatttaaaagacactgtttacaagaaaaaatgaattttgtttggac 3961ttcttttactcatgatcttgtgacatattatgtcttcatgcaaggggaaaatctcagcaa 4021tgattactctttgagatagaagaactgcaaaggtaataatacagccaaagataatctctc 4081agcttttaaatgggtagagaaacactaaagcattcaatttattcaagaaaagtaagccct 4141tgaagatatcttgaaatgaaagtataactgagttaaattatactggagaagtcttagact 4201tgaaatactacttaccatatgtgcttgcctcagtaaaatgaaccccactgggtgggcaga 4261ggttcatttcaaatacatctttgatacttgttcaaaatatgttctttaaaaatataattt 4321tttagagagctgttcccaaattttctaacgagtggaccattatcactttaaagcccttta 4381tttataatacatttcctacgggctgtgttccaacaaccattttttttcagcagactatga 4441atattatagtattataggccaaactggcaaacttcagactgaacatgtacactggtttga 4501gcttagtgaaattacttctggataattatttttttataattatggatttcaccatctttc 4561tttctgtatatatacatgtgtttttatgtaggtatatatttaccattcttcctatctatt 4621cttcctataacacacctttatcaagcatacccaggagtaatcttcaaatcttttgttata 4681ttctgaaacaaaagattgtgagtgttgcactttacctgatacacgctgatttagaaaata 4741cagaaaccatacctcactaataactttaaaatcaaagctgtgcaaagactagggggccta 4801tacttcatatgtattatgtactatgtaaaatattgactatcacacaactatttccttgga 4861tgtaattctttgttaccctttacaagtataagtgttaccttacatggaaacgaagaaaca 4921aaattcataaatttaaattcataaatttagctgaaagatactgattcaatttgtatacag 4981tgaatataaatgagacgacagcaaaattttcatgaaatgtaaaatatttttatagtttgt 5041tcatactatatgaggttctattttaaatgactttctggattttaaaaaatttctttaaat 5101acaatcatttttgtaatatttattttatgcttatgatctagataattgcagaatatcatt 5161ttatctgactctgccttcataagagagctgtggccgaattttgaacatctgttataggga 5221gtgatcaaattagaaggcaatgtggaaaaacaattctgggaaagatttctttatatgaag 5281tccctgccactagccagccatcctaattgatgaaagttatctgttcacaggcctgcagtg 5341atggtgaggaatgttctgagatttgcgaaggcatttgagtagtgaaatgtaagcacaaaa 5401cctcctgaacccagagtgtgtatacacaggaataaactttatgacatttatgtattttta 5461aaaaactttgtatcgttataaaaaggctagtcattctttcaggagaacatctaggatcat 5521agatgaaaaatcaagccccgatttagaactgtcttctccaggatggtctctaaggaaatt 5581tacatttggttctttcctactcagaactactcagaaacaactatatatttcaggttatct 5641gagcacagtgaaagcagagtactatggttgtccaacacaggcctctcagatacaagggga 5701acacaattacatattgggctagattttgcccagttcaaaatagtatttgttatcaactta 5761ctttgttacttgtatcatgaattttaaaaccctaccactttaagaagacagggatgggtt 5821attcttttttggcaggtaggctatataactatgtgattttgaaatttaactgctctggat 5881tagggagcagtgaatcaaggcagacttatgaaatctgtattatatttgtaacagaatata 5941ggaaatttaacataattgatgagctcaaatcctgaaaaatgaaagaatccaaattatttc 6001agaattatctaggttaaatattgatgtattatgatggttgcaaagtttttttgtgtgtcc 6061aataaacacattgtaaaaaaaa 10 NM_000889.2 1aaatcttccccaccctggggagtgtcacttcctcctctgccgtctcccagatcagtacac Exemplarynucleic 61aaaggctgctgctgccgccagaggaaggactgctctgcacgcacctatgtggaaactaaa acidsequence 121gcccagagagaaagtctgacttgccccacagccagtgagtgactgcagcagcaccagaat encodinghuman 181ctggtctgtttcctgtttggctcttctaccactacggcttgggatctcgggcatggtggc 7 241tttgccaatggtccttgttttgctgctggtcctgagcagaggtgagagtgaattggacgc 301caagatcccatccacaggggatgccacagaatggcggaatcctcacctgtccatgctggg 361gtcctgccagccagccccctcctgccagaagtgcatcctctcacaccccagctgtgcatg 421gtgcaagcaactgaacttcaccgcgtcgggagaggcggaggcgcggcgctgcgcccgacg 481agaggagctgctggctcgaggctgcccgctggaggagctggaggagccccgcggccagca 541ggaggtgctgcaggaccagccgctcagccagggcgcccgcggagagggtgccacccagct 601ggcgccgcagcgggtccgggtcacgctgcggcctggggagccccagcagctccaggtccg 661cttccttcgtgctgagggatacccggtggacctgtactaccttatggacctgagctactc 721catgaaggacgacctggaacgcgtgcgccagctcgggcacgctctgctggtccggctgca 781ggaagtcacccattctgtgcgcattggttttggttcctttgtggacaaaacggtgctgcc 841ctttgtgagcacagtaccctccaaactgcgccacccctgccccacccggctggagcgctg 901ccagtcaccattcagctttcaccatgtgctgtccctgacgggggacgcacaagccttcga 961gcgggaggtggggcgccagagtgtgtccggcaatctggactcgcctgaaggtggcttcga 1021tgccattctgcaggctgcactctgccaggagcagattggctggagaaatgtgtcccggct 1081gctggtgttcacttcagacgacacattccatacagctggggacgggaagttgggcggcat 1141tttcatgcccagtgatgggcactgccacttggacagcaatggcctctacagtcgcagcac 1201agagtttgactacccttctgtgggtcaggtagcccaggccctctctgcagcaaatatcca 1261gcccatctttgctgtcaccagtgccgcactgcctgtctaccaggagctgagtaaactgat 1321tcctaagtctgcagttggggagctgagtgaggactccagcaacgtggtacagctcatcat 1381ggatgcttataatagcctgtcttccaccgtgacccttgaacactcttcactccctcctgg 1441ggtccacatttcttacgaatcccagtgtgagggtcctgagaagagggagggtaaggctga 1501ggatcgaggacagtgcaaccacgtccgaatcaaccagacggtgactttctgggtttctct 1561ccaagccacccactgcctcccagagccccatctcctgaggctccgggcccttggcttctc 1621agaggagctgattgtggagttgcacacgctgtgtgactgtaattgcagtgacacccagcc 1681ccaggctccccactgcagtgatggccagggacacctacaatgtggtgtatgcagctgtgc 1741ccctggccgcctaggtcggctctgtgagtgctctgtggcagagctgtcctccccagacct 1801ggaatctgggtgccgggctcccaatggcacagggcccctgtgcagtggaaagggtcactg 1861tcaatgtggacgctgcagctgcagtggacagagctctgggcatctgtgcgagtgtgacga 1921tgccagctgtgagcgacatgagggcatcctctgcggaggctttggtcgctgccaatgtgg 1981agtatgtcactgtcatgccaaccgcacgggcagagcatgcgaatgcagtggggacatgga 2041cagttgcatcagtcccgagggagggctctgcagtgggcatggacgctgcaaatgcaaccg 2101ctgccagtgcttggacggctactatggtgctctatgcgaccaatgcccaggctgcaagac 2161accatgcgagagacaccgggactgtgcagagtgtggggccttcaggactggcccactggc 2221caccaactgcagtacagcttgtgcccataccaatgtgaccctggccttggcccctatctt 2281ggatgatggctggtgcaaagagcggaccctggacaaccagctgttcttcttcttggtgga 2341ggatgacgccagaggcacggtcgtgctcagagtgagaccccaagaaaagggagcagacca 2401cacgcaggccattgtgctgggctgcgtagggggcatcgtggcagtggggctggggctggt 2461cctggcttaccggctctcggtggaaatctatgaccgccgggaatacagtcgctttgagaa 2521ggagcagcaacaactcaactggaagcaggacagtaatcctctctacaaaagtgccatcac 2581gaccaccatcaatcctcgctttcaagaggcagacagtcccactctctgaaggagggaggg 2641acacttacccaaggctcttctccttggaggacagtgggaactggagggtgagaggaaggg 2701tgggtctgtaagaccttggtaggggactaattcactggcgaggtgcggccaccaccctac 2761ttcattttcagagtgacacccaagagggctgcttcccatgcctgcaaccttgcatccatc 2821tgggctaccccacccaagtatacaataaagtcttacctcagaccacaaaaaaaaaaaa 11 NP_000876.3 1mawearrepgprraavretvmlllclgvptgrpynvdtesallyqgphntlfgysvvlhs Exemplaryamino 61hganrwllvgaptanwlanasvinpgaiyrcrigknpgqtceqlqlgspngepcgktcle acidsequencefor 121erdnqwlgvtlsrqpgengsivtcghrwknifyiknenklptggcygvppdlrtelskri human4 181apcyqdyvkkfgenfascqagissfytkdlivmgapgssywtgslfvynittnkykafld precursor 241kqnqvkfgsylgysvgaghfrsqhttevvggapqheqigkayifsidekelnilhemkgk 301klgsyfgasvcavdlnadgfsdllvgapmqstireegrvfvyinsgsgavmnametnlvg 361sdkyaarfgesivnlgdidndgfedvaigapqeddlqgaiyiyngradgisstfsqrieg 421lqiskslsmfggsisgqidadnngyvdvavgafrsdsavllrtrpvvivdaslshpesvn 481rtkfdcvengwpsvcidltlcfsykgkevpgyivlfynmsldvnrkaespprfyfssngt 541sdvitgsiqvssreancrthqafmrkdvrdiltpiqieaayhlgphviskrsteefpplq 601pilqqkkekdimkktinfarfcahencsadlqvsakigflkphenktylavgsmktlmln 661vslfnagddayettlhvklpvglyfikileleekqincevtdnsgvvqldcsigyiyvdh 721lsridisflldvsslsraeedlsitvhatceneeemdnlkhsrvtvaiplkyevkltvhg 781fvnptsfvygsndenepetcmvekmnltfhvintgnsmapnvsveimvpnsfspqtdklf 841nildvqtttgechfenyqrvcaleqqksamqtlkgivrflsktdkrllycikadphclnf 901lcnfgkmesgkeasvhiqlegrpsilemdetsalkfeiratgfpepnprvielnkdenva 961hvlleglhhqrpkryftiviissslllglivlllisyvmwkagffkrqyksilqeenrrd 1021swsyinsksndd 12 NP_000880.1 1mvalpmvlvlllvlsrgeseldakipstgdatewrnphlsmlgscqpapscqkcilshps Exemplaryamino 61cawckqlnftasgeaearrcarreellargcpleeleeprgqqevlqdqplsqgargega acidsequencefor 121tqlapqrvrvtlrpgepqqlqvrflraegypvdlyylmdlsysmkddlervrqlghallv human7 181rlqevthsvrigfgsfvdktvlpfvstvpsklrhpcptrlercqspfsfhhvlsltgdaq precursor 241aferevgrqsvsgnldspeggfdailqaalcwewigwrnvsrllvftsddtfhtagdgkl 301ggifmpsdghchldsnglysrstefdypsvgqvaqalsaaniqpifavtsaalpvyqels 361klipksavgelsedssnvvqlimdaynslsstvtlehsslppgvhisyesqcegpekreg 421kaedrgqcnhvrinqtvtfwvslqathclpephllrlralgfseelivelhtlcdcncsd 481tqpqaphcsdgqghlqcgvcscapgrlgrlcecsvaelsspdlesgcrapngtgplcsgk 541ghcqcgrcscsgqssghlcecddascerhegilcggfgrcqcgvchchanrtgracecsg 601dmdscispegglcsghgrckcnrcqcldgyygalcdqcpgcktpcerhrdcaecgafrtg 661platncstacahtnvtlalapilddgwckertldnqlffflveddargtvvlrvrpqekg 721adhtqaivlgcvggivavglglvlayrlsveiydrreysrfekeqqqlnwkqdsnplyks 781aitttinprfgeadsptl 13 NM_016602.2 1agagatggggacggaggccacagagcaggtttcctggggccattactctggggatgaaga Exemplarynucleic 61ggacgcatactcggctgagccactgccggagctttgctacaaggccgatgtccaggcctt acidsequence 121cagccgggccttccaacccagtgtctccctgaccgtggctgcgctgggtctggccggcaa encodinghuman 181tggcctggtcctggccacccacctggcagcccgacgcgcagcgcgctcgcccacctctgc CCR10 241ccacctgctccagctggccctggccgacctcttgctggccctgactctgcccttcgcggc 301agcaggggctcttcagggctggagtctgggaagtgccacctgccgcaccatctctggcct 361ctactcggcctccttccacgccggcttcctcttcctggcctgtatcagcgccgaccgcta 421cgtggccatcgcgcgagcgctcccagccgggccgcggccctccactcccggccgcgcaca 481cttggtctccgtcatcgtgtggctgctgtcactgctcctggcgctgcctgcgctgctctt 541cagccaggatgggcagcgggaaggccaacgacgctgtcgcctcatcttccccgagggcct 601cacgcagacggtgaagggggcgagcgccgtggcgcaggtggccctgggcttcgcgctgcc 661gctgggcgtcatggtagcctgctacgcgcttctgggccgcacgctgctggccgccagggg 721gcccgagcgccggcgtgcgctgcgcgtcgtggtggctctggtggcggccttcgtggtgct 781gcagctgccctacagcctcgccctgctgctggatactgccgatctactggctgcgcgcga 841gcggagctgccctgccagcaaacgcaaggatgtcgcactgctggtgaccagcggcttggc 901cctcgcccgctgtggcctcaatcccgttctctacgccttcctgggcctgcgcttccgcca 961ggacctgcggaggctgctacggggtgggagctgcccctcagggcctcaaccccgccgcgg 1021ctgcccccgccggccccgcctttcttcctgctcagctcccacggagacccacagtctctc 1081ctgggacaactagggctgcgaatctagaggagggggcaggctgagggtcgtgggaaaggg 1141gagtaggtgggggaacactgagaaagaggcagggacctaaagggactacctctgtgcctt 1201gccacattaaattgataacatggaaatgagatgcaacccaacaa 14 AF215981.1 1agagatggggacggaggccacagagcaggtttcctggggccattactctggggatgaaga Exemplarynucleic 61ggacgcatactcggctgagccactgccggagctttgctacaaggccgatgtccaggcctt acidsequence 121cagccgggccttccaacccagtgtctccctgaccgtggctgcgctgggtctggccggcaa encodinghuman 181tggcctggtcctggccacccacctggcagcccgacgcgcagcgcgctcgcccacctctgc CCR10 241ccacctgctccagctggccctggccgacctcttgctggccctgactctgcccttcgcggc 301agcaggggctcttcagggctggagtctgggaagtgccacctgccgcaccatctctggcct 361ctactcggcctccttccacgccggcttcctcttcctggcctgtatcagcgccgaccgcta 421cgtggccatcgcgcgagcgctcccagccgggccgcggccctccactcccggccgcgcaca 481cttggtctccgtcatcgtgtggctgctgtcactgctcctggcgctgcctgcgctgctctt 541cagccaggatgggcagcgggaaggccaacgacgctgtcgcctcatcttccccgagggcct 601cacgcagacggtgaagggggcgagcgccgtggcgcaggtggccctgggcttcgcgctgcc 661gctgggcgtcatggtagcctgctacgcgcttctgggccgcacgctgctggccgccagggg 721gcccgagcgccggcgtgcgctgcgcgtcgtggtggctctggtggcggccttcgtggtgct 781gcagctgccctacagcctcgccctgctgctggatactgccgatctactggctgcgcgcga 841gcggagctgccctgccagcaaacgcaaggatgtcgcactgctggtgaccagcggcttggc 901cctcgcccgctgtggcctcaatcccgttctctacgccttcctgggcctgcgcttccgcca 961ggacctgcggaggctgctacggggtgggagctcgccctcagggcctcaaccccgccgcgg 1021ctgcccccgccggccccgcctttcttcctgctcagctcccacggagacccacagtctctc 1081ctgggacaactagggctgcgaatctagaggagggggcaggctgagggtcgtgggaaaggg 1141gagtaggtgggggaacactgagaaagaggcagggacctaaagggactacctctgtgcctt 1201gccacattaaattgataacatggaaatgaaaaaaaaaaaaaaaa 15 NP_057686.2 1mgteateqvswghysgdeedaysaeplpelcykadvqafsrafqpsvsltvaalglagng Exemplaryamino 61lvlathlaarraarsptsahllqlaladlllaltlpfaaagalqgwslgsatcrtisgly acidsequencefor 121sasfhagflflacisadryvaiaralpagprpstpgrahlvsvivwllslllalpallfs humanCCR10 181qdgqregqrrcrlifpegltqtvkgasavaqvalgfalplgvmvacyallgrtllaargp precursor 241errralrvvvalvaafvvlqlpyslallldtadllaarerscpaskrkdvallvtsglal 301arcglnpvlyaflglrfrqdlrrllrggscpsgpqprrgcprrprlsscsaptethslsw 361dn 16 P46092.3 1mgteateqvswghysgdeedaysaeplpelcykadvqafsrafqpsvsltvaalglagng Exemplaryamino 61lvlathlaarraarsptsahllqlaladlllaltlpfaaagalqgwslgsatcrtisgly acidsequencefor 121sasfhagflflacisadryvaiaralpagprpstpgrahlvsvivwllslllalpallfs humanCCR10 181qdgqregqrrcrlifpegltqtvkgasavaqvalgfalplgvmvacyallgrtllaargp precursor 241errralrvvvalvaafvvlqlpyslallldtadllaarerscpaskrkdvallvtsglal 301arcglnpvlyaflglrfrqdlrrllrggscpsgpqprrgcprrprlsscsaptethslsw 361dn 17 NM_005201.3 1tttgtagtgggaggatacctccagagaggctgctgctcattgagctgcactcacatgagg Exemplarynucleic 61atacagactttgtgaagaaggaattggcaacactgaaacctccagaacaaaggctgtcac acidsequence 121taaggtcccgctgccttgatggattatacacttgacctcagtgtgacaacagtgaccgac encodinghuman 181tactactaccctgatatcttctcaagcccctgtgatgcggaacttattcagacaaatggc CCR8 241aagttgctccttgctgtcttttattgcctcctgtttgtattcagtcttctgggaaacagc 301ctggtcatcctggtccttgtggtctgcaagaagctgaggagcatcacagatgtatacctc 361ttgaacctggccctgtctgacctgctttttgtcttctccttcccctttcagacctactat 421ctgctggaccagtgggtgtttgggactgtaatgtgcaaagtggtgtctggcttttattac 481attggcttctacagcagcatgtttttcatcaccctcatgagtgtggacaggtacctggct 541gttgtccatgccgtgtatgccctaaaggtgaggacgatcaggatgggcacaacgctgtgc 601ctggcagtatggctaaccgccattatggctaccatcccattgctagtgttttaccaagtg 661gcctctgaagatggtgttctacagtgttattcattttacaatcaacagactttgaagtgg 721aagatcttcaccaacttcaaaatgaacattttaggcttgttgatcccattcaccatcttt 781atgttctgctacattaaaatcctgcaccagctgaagaggtgtcaaaaccacaacaagacc 841aaggccatcaggttggtgctcattgtggtcattgcatctttacttttctgggtcccattc 901aacgtggttcttttcctcacttccttgcacagtatgcacatcttggatggatgtagcata 961agccaacagctgacttatgccacccatgtcacagaaatcatttcctttactcactgctgt 1021gtgaaccctgttatctatgcttttgttggggagaagttcaagaaacacctctcagaaata 1081tttcagaaaagttgcagccaaatcttcaactacctaggaagacaaatgcctagggagagc 1141tgtgaaaagtcatcatcctgccagcagcactcctcccgttcctccagcgtagactacatt 1201ttgtgaggatcaatgaagactaaatataaaaaacattttcttgaatggcatgctagtagc 1261agtgagcaaaggtgtgggtgtgaaaggtttccaaaaaaagttcagcatgaaggatgccat 1321atatgttgttgccaacacttggaacacaatgactaaagacatagttgtgcatgcctggca 1381caacatcaagcctgtgattgtgtttattgatgatgttgaacaagtggtaactttaaagga 1441ttctgtatgccaagtgaaaaaaaaagatgtctgacctccttacatat 18 BC107159.1 1ctttgtgaagaaggaattggcaacactgaaacctccagaacaaaggctgtcactaaggtc Exemplarynucleic 61ccgctgccttgatggattatacacttgacctcagtgtgacaacagtgaccgactactact acidsequence 121accctgatatcttctcaagcccctgtgatgcggaacttattcagacaaatggcaagttgc encodinghuman 181tccttgctgtcttttattgcctcctgtttgtattcagtcttctgggaaacagcctggtca CCR8 241tcctggtccttgtggtctgcaagaagctgaggagcatcacagatgtatacctcttgaacc 301tggccctgtctgacctgctttttgtcttctccttcccctttcagacctactatctgctgg 361accagtgggtgtttgggactgtaatgtgcaaagtggtgtctggcttttattacattggct 421tctacagcagcatgtttttcatcaccctcatgagtgtggacaggtacctggctgttgtcc 481atgccgtgtatgccctaaaggtgaggacgatcaggatgggcacaacgctgtgcctggcag 541tatggctaaccgccattatggctaccatcccattgctagtgttttaccaagtggcctctg 601aagatggtgttctacagtgttattcattttacaatcaacagactttgaagtggaagatct 661tcaccaacttcaaaatgaacattttaggcttgttgatcccattcaccatctttatgttct 721gctacattaaaatcctgcaccagctgaagaggtgtcaaaaccacaacaagaccaaggcca 781tcaggttggtgctcattgtggtcattgcatctttacttttctgggtcccattcaacgtgg 841ttcttttcctcacttccttgcacagtatgcacatcttggatggatgtagcataagccaac 901agctgacttatgccacccatgtcacagaaatcatttcctttactcactgctgtgtgaacc 961ctgttatctatgcttttgttggggagaagttcaagaaacacctctcagaaatatttcaga 1021aaagttgcagccaaatcttcaactacctaggaagacaaatgcctagggagagctgtgaaa 1081agtcatcatcctgccagcagcactcctcccgttcctccagcgtagactacattttgtgag 1141gatcaatgaagactaaatataaaaaacattttcttgaatggcatgctagtagcagtgagc 1201aaaggtgtgggtgtgaaaggtttccaaaaaaagttcagcatgaaggatgccgtgtgtgtt 1261gttgccaacacttggaacacgatgactggggacgtggttgtgcatgcctggcacaacatc 1321aagcctgtgattgtgtttattgatgatgttgaacaagtggtggctttggaggattctgta 1381tgccaagtgaaaggggagatgtctgacctccttcatatag 19 NP_005192.1 1mdytldlsvttvtdyyypdifsspcdaeliqtngklllavfycllfvfsllgnslvilvl Exemplaryamino 61vvckklrsitdvyllnlalsdllfvfsfpfqtyylldqwvfgtvmckvvsgfyyigfyss acidsequencefor 121mffitlmsvdrylavvhavyalkvrtirmgttlclavwltaimatipllvfyqvasedgv humanCCR8 181lqcysfynqqtlkwkiftnfkmnilgllipftifmfcyikilhqlkrcqnhnktkairlv precursor 241livviasllfwvpfnvvlfltslhsmhildgcsisqqltyathvteiisfthccvnpviy 301afvgekfkkhlseifqkscsqifnylgrqmprescekssscqqhssrsssvdyil 20 AAI07160.1 1mdytldlsvttvtdyyypdifsspcdaeliqtngklllavfycllfvfsllgnslvilvl Exemplaryamino 61vvckklrsitdvyllnlalsdllfvfsfpfqtyylldqwvfgtvmckvvsgfyyigfyss acidsequencefor 121mffitlmsvdrylavvhavyalkvrtirmgttlclavwltaimatipllvfyqvasedgv humanCCR8 181lqcysfynqqtlkwkiftnfkmnilgllipftifmfcyikilhqlkrcqnhnktkairlv precursor 241livviasllfwvpfnvvlfltslhsmhildgcsisqqltyathvteiisfthccvnpviy 301afvgekfkkhlseifqkscsqifnylgrqmprescekssscqqhssrsssvdyil 21 NM_005508.4 1gctcacaggaagccacgcacccttgaaaggcaccgggtccttcttagcatcgtgcttcct Exemplarynucleic 61gagcaagcctggcattgcctcacagaccttcctcagagccgctttcagaaaagcaagctg acidsequence 121cttctggttgggcccagacctgccttgaggagcctgtagagttaaaaaatgaaccccacg encodinghuman 181gatatagcagacaccaccctcgatgaaagcatatacagcaattactatctgtatgaaagt CCR4 241atccccaagccttgcaccaaagaaggcatcaaggcatttggggagctcttcctgccccca 301ctgtattccttggtttttgtatttggtctgcttggaaattctgtggtggttctggtcctg 361ttcaaatacaagcggctcaggtccatgactgatgtgtacctgctcaaccttgccatctcg 421gatctgctcttcgtgttttccctccctttttggggctactatgcagcagaccagtgggtt 481tttgggctaggtctgtgcaagatgatttcctggatgtacttggtgggcttttacagtggc 541atattctttgtcatgctcatgagcattgatagatacctggcaattgtgcacgcggtgttt 601tccttgagggcaaggaccttgacttatggggtcatcaccagtttggctacatggtcagtg 661gctgtgttcgcctcccttcctggctttctgttcagcacttgttatactgagcgcaaccat 721acctactgcaaaaccaagtactctctcaactccacgacgtggaaggttctcagctccctg 781gaaatcaacattctcggattggtgatccccttagggatcatgctgttttgctactccatg 841atcatcaggaccttgcagcattgtaaaaatgagaagaagaacaaggcggtgaagatgatc 901tttgccgtggtggtcctcttccttgggttctggacaccttacaacatagtgctcttccta 961gagaccctggtggagctagaagtccttcaggactgcacctttgaaagatacttggactat 1021gccatccaggccacagaaactctggcttttgttcactgctgccttaatcccatcatctac 1081ttttttctgggggagaaatttcgcaagtacatcctacagctcttcaaaacctgcaggggc 1141ctttttgtgctctgccaatactgtgggctcctccaaatttactctgctgacacccccagc 1201tcatcttacacgcagtccaccatggatcatgatctccatgatgctctgtagaaaaatgaa 1261atggtgaaatgcagagtcaatgaactttccacattcagagcttacttaaaattgtatttt 1321agtaagagattcctgagccagtgtcaggaggaaggcttacacccacagtggaaagacagc 1381ttctcatcctgcaggcagctttttctctcccactagacaagtccagcctggcaagggttc 1441acctgggctgaggcatccttcctcacaccaggcttgcctgcaggcatgagtcagtctgat 1501gagaactctgagcagtgcttgaatgaagttgtaggtaatattgcaaggcaaagactattc 1561ccttctaacctgaactgatgggtttctccagagggaattgcagagtactggctgatggag 1621taaatcgctaccttttgctgtggcaaatgggccctct 22 P51679.1 1mnptdiadttldesiysnyylyesipkpctkegikafgelflpplyslvfvfgllgnsvv Exemplaryamino 61vlvlfkykrlrsmtdvyllnlaisdllfvfslpfwgyyaadqwvfglglckmiswmylvg acidsequencefor 121fysgiffvmlmsidrylaivhavfslrartltygvitslatwsvavfaslpgflfstcyt humanCCR4 181ernhtycktkyslnsttwkvlssleinilglviplgimlfcysmiirtlqhcknekknka precursor 241vkmifavvvlflgfwtpynivlfletivelevlqdctferyldyaiqatetlafvhccln 301piiyfflgekfrkyilqlfktcrglfvlcqycgllqiysadtpsssytqstmdhdlhdal 23 NM_001206609.1 1aatcatccgagaaccttggagggtggacagtgccccttttacagatgagaaaactgaggc Exemplarynucleic 61ttgaaggggagaagcagctgcctctggcggcatggcttctggctgcaggatgcccatgga acidsequence 121gttcgtggtgaccctaggcctgtgtctcggcttcctttgctgaacttgaacaggaagatg encodinghuman 181gcagtgggggccagtggtctagaaggagataagatggctggtgccatgcctctgcaactc CLA 241ctcctgttgctgatcctactgggccctggcaacagcttgcagctgtgggacacctgggca 301gatgaagccgagaaagccttgggtcccctgcttgcccgggaccggagacaggccaccgaa 361tatgagtacctagattatgatttcctgccagaaacggagcctccagaaatgctgaggaac 421agcactgacaccactcctctgactgggcctggaacccctgagtctaccactgtggagcct 481gctgcaaggcgttctactggcctggatgcaggaggggcagtcacagagctgaccacggag 541ctggccaacatggggaacctgtccacggattcagcagctatggagatacagaccactcaa 601ccagcagccacggaggcacagaccactcaaccagtgcccacggaggcacagaccactcca 661ctggcagccacagaggcacagacaactcgactgacggccacggaggcacagaccactcca 721ctggcagccacagaggcacagaccactccaccagcagccacggaagcacagaccactcaa 781cccacaggcctggaggcacagaccactgcaccagcagccatggaggcacagaccactgca 841ccagcagccatggaagcacagaccactccaccagcagccatggaggcacagaccactcaa 901accacagccatggaggcacagaccactgcaccagaagccacggaggcacagaccactcaa 961cccacagccacggaggcacagaccactccactggcagccatggaggccctgtccacagaa 1021cccagtgccacagaggccctgtccatggaacctactaccaaaagaggtctgttcataccc 1081ttttctgtgtcctctgttactcacaagggcattcccatggcagccagcaatttgtccgtc 1141aactacccagtgggggccccagaccacatctctgtgaagcagtgcctgctggccatccta 1201atcttggcgctggtggccactatcttcttcgtgtgcactgtggtgctggcggtccgcctc 1261tcccgcaagggccacatgtaccccgtgcgtaattactcccccaccgagatggtctgcatc 1321tcatccctgttgcctgatgggggtgaggggccctctgccacagccaatgggggcctgtcc 1381aaggccaagagcccgggcctgacgccagagcccagggaggaccgtgagggggatgacctc 1441accctgcacagcttcctcccttagctcactctgccatctgttttggcaagaccccacctc 1501cacgggctctcctgggccacccctgagtgcccagaccccattccacagctctgggcttcc 1561tcggagacccctggggatggggatcttcagggaaggaactctggccacccaaacaggaca 1621agagcagcctggggccaagcagacgggcaagtggagccacctctttcctccctccgcgga 1681tgaagcccagccacatttcagccgaggtccaaggcaggaggccatttacttgagacagat 1741tctctcctttttcctgtcccccatcttctctgggtccctctaacatctcccatggctctc 1801cccgcttctcctggtcactggagtctcctccccatgtacccaaggaagatggagctcccc 1861catcccacacgcactgcactgccattgtcttttggttgccatggtcaccaaacaggaagt 1921ggacattctaagggaggagtactgaagagtgacggacttctgaggctgtttcctgctgct 1981cctctgacttggggcagcttgggtcttcttgggcacctctctgggaaaacccagggtgag 2041gttcagcctgtgagggctgggatgggtttcgtgggcccaagggcagacctttctttggga 2101ctgtgtggaccaaggagcttccatctagtgacaagtgacccccagctatcgcctcttgcc 2161ttcccctgtggccactttccagggtggactctgtcttgttcactgcagtatcccaactgc 2221aggtccagtgcaggcaataaatatgtgatggacaaacgatagcggaatccttcaaggttt 2281caaggctgtctccttcaggcagccttcccggaattctccatccctcagtgcaggatgggg 2341gctggtcctcagctgtctgccctcagcccctggccccccaggaagcctctttcatgggct 2401gttaggttgacttcagttttgcctcttggacaacagggggtcttgtacatccttgggtga 2461ccaggaaaagttcaggctatggggggccaaagggagggctgccccttccccaccagtgac 2521cactttattccacttcctccattacccagttttggcccacagagtttggtcccccccaaa 2581cctcggaccaatatccctctaaacatcaatctatcctcctgttaaagaaaaaaaaaaa 24 NM_003006.4 1acacacagccattgggggttgctcggatccgggactgccgcagggggtgccacagcagtg Exemplarynucleic 61cctggcagcgtgggctgggaccttgtcactaaagcagagaagccacttcttctgggccca acidsequence 121cgaggcagctgtcccatgctctgctgagcacggtggtgccatgcctctgcaactcctcct encodinghuman 181gttgctgatcctactgggccctggcaacagcttgcagctgtgggacacctgggcagatga CLA 241agccgagaaagccttgggtcccctgcttgcccgggaccggagacaggccaccgaatatga 301gtacctagattatgatttcctgccagaaacggagcctccagaaatgctgaggaacagcac 361tgacaccactcctctgactgggcctggaacccctgagtctaccactgtggagcctgctgc 421aaggcgttctactggcctggatgcaggaggggcagtcacagagctgaccacggagctggc 481caacatggggaacctgtccacggattcagcagctatggagatacagaccactcaaccagc 541agccacggaggcacagaccactcaaccagtgcccacggaggcacagaccactccactggc 601agccacagaggcacagacaactcgactgacggccacggaggcacagaccactccactggc 661agccacagaggcacagaccactccaccagcagccacggaagcacagaccactcaacccac 721aggcctggaggcacagaccactgcaccagcagccatggaggcacagaccactgcaccagc 781agccatggaagcacagaccactccaccagcagccatggaggcacagaccactcaaaccac 841agccatggaggcacagaccactgcaccagaagccacggaggcacagaccactcaacccac 901agccacggaggcacagaccactccactggcagccatggaggccctgtccacagaacccag 961tgccacagaggccctgtccatggaacctactaccaaaagaggtctgttcatacccttttc 1021tgtgtcctctgttactcacaagggcattcccatggcagccagcaatttgtccgtcaacta 1081cccagtgggggccccagaccacatctctgtgaagcagtgcctgctggccatcctaatctt 1141ggcgctggtggccactatcttcttcgtgtgcactgtggtgctggcggtccgcctctcccg 1201caagggccacatgtaccccgtgcgtaattactcccccaccgagatggtctgcatctcatc 1261cctgttgcctgatgggggtgaggggccctctgccacagccaatgggggcctgtccaaggc 1321caagagcccgggcctgacgccagagcccagggaggaccgtgagggggatgacctcaccct 1381gcacagcttcctcccttagctcactctgccatctgttttggcaagaccccacctccacgg 1441gctctcctgggccacccctgagtgcccagaccccattccacagctctgggcttcctcgga 1501gacccctggggatggggatcttcagggaaggaactctggccacccaaacaggacaagagc 1561agcctggggccaagcagacgggcaagtggagccacctctttcctccctccgcggatgaag 1621cccagccacatttcagccgaggtccaaggcaggaggccatttacttgagacagattctct 1681cctttttcctgtcccccatcttctctgggtccctctaacatctcccatggctctccccgc 1741ttctcctggtcactggagtctcctccccatgtacccaaggaagatggagctcccccatcc 1801cacacgcactgcactgccattgtcttttggttgccatggtcaccaaacaggaagtggaca 1861ttctaagggaggagtactgaagagtgacggacttctgaggctgtttcctgctgctcctct 1921gacttggggcagcttgggtcttcttgggcacctctctgggaaaacccagggtgaggttca 1981gcctgtgagggctgggatgggtttcgtgggcccaagggcagacctttctttgggactgtg 2041tggaccaaggagcttccatctagtgacaagtgacccccagctatcgcctcttgccttccc 2101ctgtggccactttccagggtggactctgtcttgttcactgcagtatcccaactgcaggtc 2161cagtgcaggcaataaatatgtgatggacaaacgatagcggaatccttcaaggtttcaagg 2221ctgtctccttcaggcagccttcccggaattctccatccctcagtgcaggatgggggctgg 2281tcctcagctgtctgccctcagcccctggccccccaggaagcctctttcatgggctgttag 2341gttgacttcagttttgcctcttggacaacagggggtcttgtacatccttgggtgaccagg 2401aaaagttcaggctatggggggccaaagggagggctgccccttccccaccagtgaccactt 2461tattccacttcctccattacccagttttggcccacagagtttggtcccccccaaacctcg 2521gaccaatatccctctaaacatcaatctatcctcctgttaaagaaaaaaaaaaa 25 NP_001193538.1 1mavgasglegdkmagamplqlllllillgpgnslqlwdtwadeaekalgpllardrrqat Exemplaryamino 61eyeyldydflpeteppemlrnstdttpltgpgtpesttvepaarrstgldaggavteltt acidsequence 121elanmgnlstdsaameiqttgpaateaqttqpvpteaqttplaateaqttrltateaqtt forhumanCLA 181plaateaqttppaateaqttqptgleaqttapaameaqttapaameaqttppaameaqtt precursor 241qttameaqttapeateaqttqptateaqttplaamealstepsatealsmepttkrglfi 301pfsyssvthkgipmaasnlsvnypvgapdhisvkqcllaililalvatiffvctvvlavr 361lsrkghmypvrnysptemvcissllpdggegpsatangglskakspgltpepredregdd 421ltlhsflp 26 NP_002997.2 1mplqlllllillgpgnslqlwdtwadeaekalgpllardrrqateyeyldydflpetepp Exemplaryamino 61emlrnstdttpltgpgtpesttvepaarrstgldaggavtelttelanmgnlstdsaame acidsequence 121iqttgpaateaqttqpvpteaqttplaateaqttrltateaqttplaateaqttppaate forhumanCLA 181aqttqptgleaqttapaameaqttapaameaqttppaameaqttqttameaqttapeate precursor 241aqttqptateaqttplaamealstepsatealsmepttkrglfipfsvssvthkgipmaa 301snlsvnypvgapdhisvkqcllaililalvatiffvctvvlavrlsrkghmypvrnyspt 361emvcissllpdggegpsatangglskakspgltpepredregddltlhsflp
[0046] 4.4. Chimeric Antigen Receptors
[0047] When the cells provided herein are T lymphocytes which comprise homing receptors, as described above, such T lymphocytes can, in certain embodiments, comprise chimeric antigen receptors (CARs), which are artificial membrane-bound proteins that direct a T lymphocyte to an antigen, and stimulate the T lymphocyte to kill a cell displaying the antigen. See, e.g., Eshhar, U.S. Pat. No. 7,741,465. At a minimum, the CAR comprises an extracellular domain that binds to an antigen, e.g., an antigen on a cell, a transmembrane domain, and an intracellular (cytoplasmic) signaling domain that transmits a primary activation signal to an immune cell. All other conditions being satisfied, when the CAR is expressed on the surface of, e.g., a T lymphocyte, for example, a primary T lymphocyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cell surface, to kill the cell expressing the antigen. Because T lymphocytes require two signals, a primary activation signal and a costimulatory signal, in order to maximally activate, typically CARs also comprise a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal.
[0048] 4.4.1. General CAR Structure Intracellular Domain
[0049] In certain embodiments, the intracellular domain of the CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of T lymphocytes and triggers activation and/or proliferation of said T lymphocytes. Such a domain or motif is able to transmit a primary antigen-binding signal that is necessary for the activation of a T lymphocyte in response to the antigen's binding to the CAR's extracellular portion. Typically, this domain or motif comprises, or is, an ITAM (immunoreceptor tyrosine-based activation motif). ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is a CD3 intracellular signaling domain. In other specific embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.
[0050] In certain embodiments, the CAR additionally comprises one or more co-stimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide. The one or more co-stimulatory domains or motifs can be, or comprise, one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domain or motif.
[0051] The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, typically a transmembrane region from a CD4 or a CD8 molecule.
[0052] 4.4.2. CAR Extracellular Domain
[0053] The extracellular domain of the polypeptide binds to an antigen of interest. In certain embodiments of any of the polypeptides described herein, the extracellular domain comprises a receptor, or a portion of a receptor, that binds to said antigen. The extracellular domain may be, e.g., a receptor, or a portion of a receptor, that binds to said antigen. In certain embodiments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof. In specific embodiments, the extracellular domain comprises, or is, a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a V.sub.L linked to V.sub.H by a flexible linker, wherein said V.sub.L and V.sub.H are from an antibody that binds said antigen.
[0054] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, e.g., can be an antigen on a tumor cell. The tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer. The antigen can be any antigen that is expressed on a cell of any tumor or cancer type, e.g., cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like. In more specific embodiments, said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrm macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.
[0055] In certain embodiments, the antigen is a tumor-associated antigen or a tumor-specific antigen. In various specific embodiments, without limitation, the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein.
[0056] In certain embodiments, the TAA or TSA is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.
[0057] In certain other embodiments, the TAA or TSA is a carbohydrate or ganglioside, e.g., fuc-GM1, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.
[0058] In certain other embodiments, the TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor-specific antigen is integrin v3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those in the art.
[0059] Antibodies, and scFvs, that bind to TSAs and TAAs are known in the art, as are nucleotide sequences that encode them.
[0060] In certain specific embodiments, the antigen is an antigen not considered to be a TSA or a TAA, but which is nevertheless associated with tumor cells, or damage caused by a tumor. In certain embodiments, for example, the antigen is, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins can include, e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a hypoxic environment local to the tumor. As such, in other specific embodiments, the antigen is a hypoxia-associated factor, e.g., HIF-1, HIF-1, HIF-2, HIF-2, HIF-3, or HIF-3. Tumors can also cause localized damage to normal tissue, causing the release of molecules known as damage associated molecular pattern molecules (DAMPs; also known as alarmins). In certain other specific embodiments, therefore, the antigen is a DAMP, e.g., a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be a deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
[0061] In a specific embodiment, in which the cancer a gastrointestinal cancer, for example, liver cancer, stomach cancer, esophageal cancer, gallbladder cancer, colorectal cancer, anal cancer, or pancreatic cancer, the antigen is an antigen specific for or associated with a gastrointestinal cancer. In a specific embodiment, T lymphocytes provided herein express a gastrointestinal homing receptor and also express a CAR with an extracellular domain that binds to an antigen associated with a gastrointestinal cancer. In a specific embodiment, the extracellular domain of the CAR binds CEA. In other specific embodiments, the extracellular domain of the CAR binds Her2, CA242, MUC1, CA125, or CA19-9.
[0062] In a specific embodiment, in which the cancer is a skin cancer, for example, melanoma, squamous cell carcinoma, or basal cell carcinoma, the antigen is an antigen specific for or associated with a skin cancer. In a specific embodiment, T lymphocytes provided herein express a skin homing receptor and also express a CAR with an extracellular domain that binds to an antigen associated with a skin cancer. In a specific embodiment, the extracellular domain of the CAR binds HMW-MAA. In other specific embodiments, the extracellular domain of the CAR binds Her2, GD2, GD3, CEA, or SPAG9.
[0063] In certain embodiments of the polypeptides described herein, the extracellular domain is joined to said transmembrane domain by a linker, spacer or hinge polypeptide sequence, e.g., a sequence from CD28.
[0064] 4.5. Isolated Nucleic Acids
[0065] In one embodiment, provided herein are polynucleotide sequences that encode the polypeptides provided herein (e.g., chimeric receptors and homing receptors). The polynucleotides may be contained within any polynucleotide vector suitable for the transformation of immune cells, e.g., T lymphocytes. For example, T lymphocytes may be transformed using synthetic vectors, lentiviral or retroviral vectors, autonomously replicating plasmids, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or the like, containing polynucleotides encoding the first and second polypeptides (e.g., chimeric receptors). Lentiviral vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the lentiviral vectors described in U.S. Pat. Nos. 5,994,136; 6,165,782; 6,428,953; 7,083,981; and 7,250,299, the disclosures of which are hereby incorporated by reference in their entireties. HIV vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the vectors described in U.S. Pat. No. 5,665,577, the disclosure of which is hereby incorporated by reference in its entirety.
[0066] Nucleic acids useful in the production of the polypeptides provided herein, e.g., within a T lymphocyte, include DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone, and can include deoxyuridine substitution for deoxythymidine, 5-methyl-2-deoxycytidine or 5-bromo-2-deoxycytidine substitution for deoxycytidine. Modifications of the sugar moiety can include modification of the 2 hydroxyl of the ribose sugar to form 2-O-methyl or 2-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chain. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
[0067] A nucleic acid encoding a polypeptide provided herein may be introduced into host cells as part of a vector, such as, e.g., an expression vector. In addition, a polypeptide provided herein may be produced by transfecting a host cell with a nucleic acid encoding such a polypeptide, and such nucleic acid may be part of a vector. In a specific embodiment, the vector is an expression vector that is capable of directing the expression of a nucleic acid encoding a polypeptide provided herein. Non-limiting examples of expression vectors include, but are not limited to, plasmids and viral vectors, such as replication defective retroviruses, adenoviruses, adeno-associated viruses, Newcastle disease virus, vaccinia virus and baculoviruses. Standard molecular biology techniques may be used to introduce a nucleic acid encoding a polypeptide provided herein into an expression vector.
[0068] An expression vector comprises a nucleic acid encoding a polypeptide provided herein in a form suitable for expression of the nucleic acid in a host cell or non-human subject. In a specific embodiment, an expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid to be expressed. Within an expression vector, operably linked is intended to mean that a nucleic acid of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleic acid in many types of host cells, those which direct expression of the nucleic acid only in certain host cells (e.g., tissue-specific regulatory sequences), and those which direct the expression of the nucleic acid upon stimulation with a particular agent (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as, e.g., the choice of the host cell to be transformed, the level of expression of protein desired, etc.
[0069] An expression vector can be introduced into host cells via conventional transformation or transfection techniques. Such techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., 1989, Molecular CloningA Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, and other laboratory manuals. In certain embodiments, a host cell is transiently transfected with an expression vector containing a nucleic acid encoding a polypeptide provided herein. In other embodiments, a host cell is stably transfected with an expression vector containing a nucleic acid encoding a polypeptide provided herein.
[0070] 4.6. Cells
[0071] Non-limiting examples of cells in which the homing receptors may be used include, but are not limited to, natural killer (NK) cells, dendritic cells (DC), placental stem cells (e.g., the placental stem cells disclosed in U.S. Pat. Nos. 7,468,276; 8,057,788 and 8,202,703, the disclosures of which are hereby incorporated by reference in their entireties), mesenchymal-like stem cells from umbilical cord blood, placental blood, peripheral blood, bone marrow, dental pulp, adipose tissue, osteochondral tissue, and the like; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (e.g., fibroblasts, etc.). The homing receptors may also be used in tumor cell lines, e.g., for animal model experimental purposes.
[0072] In a specific embodiment, the cells comprising the polypeptides provided herein are T lymphocytes. The T lymphocytes comprising the polypeptides provided herein may be naive T lymphocytes or MHC-restricted T lymphocytes. In certain embodiments, the T lymphocytes are tumor infiltrating lymphocytes (TILs). In certain embodiments, the T lymphocytes have been isolated from a tumor biopsy, or have been expanded from T lymphocytes isolated from a tumor biopsy. In certain other embodiments, the T lymphocytes have been isolated from, or are expanded from T lymphocytes expanded from, peripheral blood, cord blood, or lymph.
[0073] In certain embodiments, the immune cells, e.g., T lymphocytes, used in the present methods are autologous to an individual to whom the T lymphocytes are to be administered. In certain embodiments, the T lymphocytes are allogeneic to an individual to whom the T lymphocytes are to be administered. Where allogeneic T lymphocytes are used to prepare T lymphocytes, it is preferable to select T lymphocytes that will reduce the possibility of graft-versus-host disease (GVHD) in the individual. For example, in certain embodiments, virus-specific T lymphocytes are selected for preparation of T lymphocytes; such lymphocytes will be expected to have a greatly reduced native capacity to bind to, and thus become activated by, any recipient antigens. In certain embodiments, recipient-mediated rejection of allogeneic T lymphocytes can be reduced by co-administration to the host of one or more immunosuppressive agents, e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.
[0074] In one embodiment, T lymphocytes are obtained from an individual, optionally then expanded, and then transformed with a polynucleotide encoding one or more homing receptors, and optionally one or more polynucleotides encoding one or more CAR(s), and optionally then expanded. In another embodiment, T lymphocytes are obtained from an individual, optionally then expanded, and then transformed with a polynucleotide encoding one or more homing receptors, and optionally one or more polynucleotides encoding one or more CAR(s), and optionally then expanded. Cells containing any of the polynucleotide may be selected using one or more selectable markers.
[0075] In certain embodiments, any of the T lymphocytes provided herein express or comprise native TCR proteins, e.g., TCR- and TCR- that are capable of forming native TCR complexes. In certain other embodiments, either or both of the native genes encoding TCR- and TCR- in the T lymphocytes are modified to be non-functional, e.g., a portion or all are deleted, a mutation is inserted, etc.
[0076] In certain embodiments, any of the T lymphocytes provided herein are isolated from a tumor lesion, e.g., are tumor-infiltrating lymphocytes; such T lymphocytes are expected to be specific for a TSA or TAA.
[0077] T lymphocytes, and T lymphocytes comprising a polypeptide comprising a CD3 signaling domain and a CD28 co-stimulatory domain can be expanded using antibodies to CD3 and CD28, e.g., antibodies attached to beads, or to the surface of a cell culture plate; see, e.g., U.S. Pat. Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681.
[0078] In certain embodiments, the antigen and/or antibody can exist free in the medium in which the T lymphocytes are cultured, or either or both can be attached to a solid support, e.g., tissue culture plastic surface, beads, or the like.
[0079] 4.7. Methods of Using Cells Comprising Homing Receptors
[0080] In one aspect, the cells, e.g., T lymphocytes, provided herein that comprise one or more homing receptors and optionally one or more CARs, as described elsewhere herein, are used to treat an individual having one or more types of cells desired to be targeted by the cells described herein, e.g., one or more types of cells to be killed. In certain embodiments, the cells to be killed are cancer cells, e.g., tumor cells. In specific embodiments, the cancer cells are cells of a solid tumor. In specific embodiments, the cells are cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like. In more specific embodiments, said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrm macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.
[0081] In certain embodiments, the modified cells, e.g., modified T lymphocytes described herein are administered to a subject in need thereof, such that the combination of homing receptors selected is compatible with the patient population (or subpopulation) in which the cells, e.g., T lymphocytes, have been administered. For example, the combination of homing receptors is chosen based on the type of tumor or cancer present in the patient.
[0082] In specific embodiments, T lymphocytes expressing gastrointestinal homing receptors are administered to patients having a gastrointestinal tumor or cancer. In certain embodiments, the gastrointestinal tumor or cancer is esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, or anal cancer. In a specific embodiment, said T cells further comprise a CAR with an extracellular domain that binds to an antigen associated with a gastrointestinal tumor or cancer.
[0083] In other specific embodiments, T lymphocytes expressing skin homing receptors are administered to patients having a skin tumor or cancer. In certain embodiments, the skin tumor or cancer is melanoma, basal cell carcinoma, or squamous cell carcinoma. In a specific embodiment, said T cells further comprise a CAR with an extracellular domain that binds to an antigen associated with a skin tumor or cancer. In specific embodiments, the T lymphocytes comprising a homing receptor also comprise a CAR with an extracellular domain that binds to an antigen expressed in the tumor or cancer cells of the patient.
[0084] Efficacy of the cells, e.g., T lymphocytes, after administration to an individual having a disease or disorder remediable by such cells, e.g., T lymphocytes, e.g., an individual having cancer, can be assessed by one or more criteria, specific to the particular disease or disorder, known to those of ordinary skill in the art, to be indicative of progress of the disease or disorder. Generally, administration of the cells to such an individual is effective when one or more of said criteria detectably, e.g., significantly, moves from a disease state value or range to, or towards, a normal value or range.
[0085] In certain embodiments, the cells, e.g., T lymphocytes, are formulated in a pharmaceutically-acceptable solution. In preferred embodiments, the pharmaceutically-acceptable solution is suitable for the delivery of living cells. In specific embodiments, the pharmaceutically-acceptable solution is, for example, saline solution (such as Ringer's solution), gelatins, carbohydrates (e.g., lactose, amylose, starch, or the like), fatty acid esters, hydroxymethylcellulose, or polyvinyl pyrolidine. In more specific embodiments, the pharmaceutically-acceptable solution is sterilized prior to addition of the cells. In other more specific embodiments, the pharmaceutically-acceptable solution may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical carriers suitable for use in formulating the cells are known in the art and are described, for example, in WO 96/05309.
[0086] In certain embodiments, the cells, e.g., T lymphocytes, are formulated into individual doses, wherein said individual doses comprise at least, at most, or about 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6, 510.sup.6, 110.sup.7, 510.sup.7, 110.sup.8, 510.sup.8, 110.sup.9, 510.sup.9, 110.sup.10, 510.sup.10, or 110.sup.11 T lymphocytes. In certain embodiments, the cells, e.g., T lymphocytes, are formulated into individual doses, wherein said individual does comprise a range from 110.sup.4 to 510.sup.4, 510.sup.4 to 110.sup.5, 110.sup.5 to 510.sup.5, 510.sup.5 to 110.sup.6 110.sup.6 to 510.sup.6, 510.sup.6 to 110.sup.7, 110.sup.7 to 510.sup.7, 510.sup.7 to 110.sup.8 110.sup.8 to 510.sup.8, 510.sup.8 to 110.sup.9, 110.sup.9 to 510.sup.9, 510.sup.9 to 110.sup.10, 110.sup.10 to 510.sup.10, or 510.sup.10 to 110.sup.11 T lymphocytes. In certain embodiments, the cells are formulated for intravenous, intra-arterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or administration within a particular organ or tissue.
5. EXAMPLES
5.1. Example 1: Generation of Murine CXCR5.SUP.+ CAR T Cells and In Vivo Study
[0087] T lymphocytes are obtained from the spleen of B6 Thy 1.1 mice. A lentiviral construct comprising a chimeric antigen receptor (CAR) comprising humanized mouse anti-human CEA-scFv or anti-human HER2-scFv and mouse co-stimulation intracellular domain and CD3 is generated. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a mouse CXCL13 chemotaxis assay.
[0088] For the in vivo assay, the activities of murine CXCR5+ CAR T cells are compared to the activities of CXCR5 CAR T cells and non-transduced T cells. Human CEA (or HER2)-transgenic mice that can spontaneously develop CEA+ gastrointestinal tumors (or HER2+ mammary tumors) are used as subjects. Adoptive transfer of the cells is accomplished with one or more doses by intravenous administration.
[0089] To examine the location of adoptively transferred CAR T cells in draining lymph nodes, (mesenteric lymph nodes for CEA transgenic mice or axillary lymph nodes for HER2 transgenic mice), a readout assay is done via draining lymph node immunohistochemistry. Animals are sacrificed and the draining lymph nodes are identified. Lymph nodes are grasped with curved forceps and pulled free of attached tissue. Lymph nodes are embedded in optimal cutting temperature compound and frozen on dry ice, and then stored at 80 C. Cryosections are cut, air-dried, and fixed in acetone. Sections are pre-incubated with rabbit (or goat) serum at room temperature, and subsequently incubated with primary antibodies (rat anti-mouse CD3, CD8, CD4, biotinylated mouse anti-mouse Thy-1.1, biotinylated goat-anti-human IgG (H+L)), and biotinylated rabbit (or goat) anti-rat IgG (mouse adsorbed) followed by avidin-peroxidase (or fluorochrome) conjugate. Then sections are incubated with substrate of peroxidase for color development. The images of immunohistochemistry staining results are acquired using confocal microscopy. Donor T cells are identified via anti-mouse Thy1.1 and CAR T cells are identified with anti-human IgG.
[0090] To examine the antigen-specific immune response establishment in the draining lymph nodes, a readout assay is done via flow cytometry of CAR T cells isolated from draining lymph nodes. Phenotypic characterization of surface markers Thy 1.1, anti-human IgG, CXCR5, CD69, and HLA-DR is performed. CD69 is a marker for activated T cells. HLA-DR is a marker for antigen-presenting cells. Functional evaluation is done to assay proliferation, Ki67 expression, and numerization of CAR T cells. Cytokine production is evaluated by intracellular cytokine staining upon stimulation.
[0091] To examine tumor killing activity of CAR T cells in a tumor site, tumor volume and survival are assayed. Volumes of tumors are determined in vivo by external caliper. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) are determined. Tumor volume based on caliper measurements are calculated by the modified ellipsoidal formula: tumor volume=(lengthwidth).
5.2. Example 2: Generation of Human CXCR5.SUP.+ CAR T Cells
[0092] T lymphocytes are obtained from human PBMCs. A lentiviral construct comprising a chimeric antigen receptor (CAR) composed of humanized mouse anti-human CEA-scFv or anti-human HER2-scFv and human co-stimulation intracellular domain and CD3 is generated and used to transduce the T cells to create a population of CEA-specific CAR T cells. The CAR T cells are further transduced with lentiviral vectors containing nucleic acid sequences encoding human CXCR5; CAR T cells expressing CXCR5 are selected for further study. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a human CXCL13 chemotaxis assay.
5.3. Example 3: Generation of Murine Gastrointestinal Homing CAR T Cells and in Vivo Study
[0093] T lymphocytes are obtained from the spleen of mice. CAR T cells are generated using a lentiviral construct, as described above, containing a nucleotide sequence encoding a CAR comprising a humanized mouse anti-human CEA-scFv and mouse co-stimulation intracellular domain and CD3. The gastrointestinal homing receptors are introduced via lentiviral transduction of mouse CCR9 and 47 nucleic acid sequences. Retinoic acid treatment is performed during T cell activation. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a mouse TECK (thymus expressed chemokine, or CCL25) chemotaxis assay (Binger, et al., 2009, Experimental Cell Research 315:1468-79). The chemotactic response to TECK is assessed using 5 m pore-size polycarbonate membranes in 96-multiwell format chemotaxis plates. Lower wells containing TECK in deprivation medium are separated from the upper wells by the membrane. The T cells, in deprivation medium, are seeded into the upper wells and incubated at 37 C. for 20 hours. Non-migrated cells left on top of the membrane are removed. Migrated cells are fixed, stained, and enumerated microscopically or are enumerated via flow cytometry with counting beads. Negative controls are performed with deprivation medium without chemokine, and positive controls are performed with medium for cell culturing containing 10% FBS.
[0094] For an in vivo assay, human CEA-transgenic mice that can spontaneously develop CEA+ gastrointestinal tumors are used as subjects. Adoptive transfer of the cells is accomplished with one or more doses by intravenous administration.
[0095] To examine the location of adoptively transferred CAR T cells in established gastrointestinal tract tumors, a readout assay is done via gastrointestinal tract immunohistochemistry. Animals are sacrificed and the gastrointestinal tracts are removed. The small intestine is sectioned into 4-5 parts before immersion fixation. Sections are pre-incubated with rabbit (or goat) serum at room temperature, and subsequently incubated with primary antibodies (rat anti-mouse CD3, biotinylated mouse anti-mouse Thy-1.1, biotinylated goat-anti-human IgG (H+L)), and biotinylated rabbit (or goat) anti-rat IgG (mouse adsorbed) followed by avidin-peroxidase (or fluorochrome) conjugate. Then sections are incubated with substrate of peroxidase for color development. The images of immunohistochemistry staining results are acquired using microscopy. Donor T cells are identified via anti-mouse Thy1.1 and CAR T cells are identified with anti-human IgG. qRT-PCR or PCR of gastrointestinal tract for detection of migrated CAR T cells in the tissue is also performed.
[0096] To examine the antigen-specific immune response establishment in the gastrointestinal tract, a readout assay is done via flow cytometry of CAR T cells isolated from the gastrointestinal tract. Phenotypic characterization of surface markers Thy 1.1, anti-human IgG, CCR9, 47, CD69, and HLA-DR is performed. Functional evaluation is done to assay proliferation, Ki67 expression, and numerization of CAR T cells. Cytokine production is evaluated by intracellular cytokine staining upon stimulation.
[0097] To examine tumor killing activity of CAR T cells in a tumor site, tumor volume and survival are assayed.
[0098] 5.4. Example 4: Generation of Human Gastrointestinal Homing CAR T Cells
[0099] T lymphocytes are obtained from human PBMCs. Generation of CAR T cells is performed using a CAR comprising humanized mouse anti-human CEA-scFv and mouse co-stimulation intracellular domain and CD3. The CAR T cells are further transduced with lentiviral vectors containing nucleic acid sequences encoding human CCR9 and 47; CAR T cells expressing both of these proteins are selected for further study. During subsequent T cell activation, the CAR T cells are contacted with retinoic acid such that the level of the CCR9 and 47 are increased as compared to CAR T cells expressing these two proteins, but not contacted with retinoic acid. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a human TECK chemotaxis assay.
5.5. Example 5: Generation of Mouse Skin Homing CAR T Cells and In Vivo Study
[0100] T lymphocytes are obtained from mouse spleen. Generation of CAR T cells is performed using a CAR comprising humanized mouse anti-human HMW-MAA-scFv and mouse co-stimulation intracellular domain and CD3. The CAR T cell s are further transduced with lentiviral vectors containing nucleic acid sequences encoding human CCR10 or CCR4, and/or CLA; CAR T cells expressing both of these proteins are selected for further study. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a mouse CCL27 chemotaxis assay. The chemotactic response to CCL27 is assessed using 5 m pore-size polycarbonate membranes in 96-multiwell format chemotaxis plates. Lower wells containing CCL27 in deprivation medium are separated from the upper wells by the membrane. The T cells, in deprivation medium, are seeded into the upper wells and incubated at 37 C. for 20 hours. Non-migrated cells left on top of the membrane are removed. Migrated cells are fixed, stained, and enumerated microscopically or are enumerated via flow cytometry with counting beads. Negative controls are performed with deprivation medium without chemokine, and positive controls are performed with medium for cell culturing containing 10% FBS.
[0101] For the in vivo assay, immunodeficient mice engrafted with human melanoma cell line that expresses HMW-MAA are used as subjects. Cells are administered through adoptive transfer with multiple doses of intravenous administration.
[0102] To examine the location of adoptively transferred CAR T cells in established melanoma, a readout assay is done via immunohistochemistry. Donor T cells are identified via anti-CD3 and CAR T cells are identified with anti-mouse IgG. qRT-PCR or PCR for detection of migrated CAR T cells in the tissue is also performed.
[0103] To examine the antigen-specific immune response establishment in the melanoma, a readout assay is done via flow cytometry of CAR T cells isolated from the melanoma. Phenotypic characterization of surface markers CD3, anti-mouse IgG, CCR10 (or CCR4), CLA, CD69, and HLA-DR is performed. Functional evaluation is done to assay proliferation, Ki67 expression, and numerization of CAR T cells. Cytokine production is evaluated by intracellular cytokine staining upon stimulation.
[0104] To examine tumor killing activity of CAR T cells in a tumor site, tumor volume and survival are assayed.
5.6. Example 6: Generation of Human Skin Homing CAR T Cells
[0105] T lymphocytes are obtained from human PBMCs. CAR T cells generation is performed using a CAR composed of humanized mouse anti-human HMW-MAA-scFv and human co-stimulation intracellular domain and CD3. The gastrointestinal homing receptors are introduced via lentiviral transduction of human CCR10 or CCR4, and CLA nucleic acid sequences. Treatment with 1,25(OH).sub.2VD.sub.3 and IL-12 is performed during T cell activation. Validation of the CAR T cells is done via phenotypic characterization and functional evaluation via a human CCL27 chemotaxis assay, as in Example 5.
5.7. Example 7: Treatment of Gastrointestinal Tumor
[0106] An individual presents with a gastrointestinal tumor, for example, adenomatous polyposis coli. Testing of tumor cells from the individual determines that the tumor cells express CEA. T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence that encodes a chimeric antigen receptor (CAR), and transfected with a second lentiviral vector comprising a nucleotide sequence encoding human CCR9 and/or 47 nucleic acid sequences. The T lymphocytes are expanded using CD3+CD28-coated beads in the presence of retinoic acid to sufficient numbers for administration. The chimeric receptor comprises an extracellular antigen-binding region that binds to CEA; a transmembrane domain; an intracellular co-stimulatory domain from CD28; and an intracellular CD3 domain. The individual is administered between 10.sup.9 and 10.sup.10 of the T lymphocytes in 200 mL saline solution by intravenous infusion over 30 minutes. The individual is re-assessed for the gastrointestinal tumor stage and spread to lymph nodes, and histology of biopsied gastrointestinal tissue is performed at 30, 60, and 90 days post-administration.
5.8. Example 8: Treatment of Melanoma
[0107] An individual presents with melanoma. Testing of tumor cells from the individual determines that the tumor cells express HMW-MAA. T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence that encodes a chimeric antigen receptor (CAR), and transfected with a second lentiviral vector comprising a nucleotide sequence encoding human CCR10 (or CCR4) and CLA nucleic acid sequences. The T lymphocytes are expanded using CD3+CD28-coated beads in the presence of 1,25(OH).sub.2VD.sub.3 and IL-12 to sufficient numbers for administration. The chimeric receptor comprises an extracellular antigen-binding region that binds to HMW-MAA; a transmembrane domain; an intracellular co-stimulatory domain from CD28; and an intracellular CD3 domain. The individual is administered between 10.sup.9 and 10.sup.10 of the T lymphocytes in 200 mL saline solution by intravenous infusion over 30 minutes. The individual is re-assessed for the melanoma stage and spread to lymph nodes, and histology of biopsied skin tissue is performed at 30, 60, and 90 days post-administration.
EQUIVALENTS
[0108] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
[0109] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.
[0110] The T lymphocytes of the invention are for use in the methods disclosed herein