Methods and compositions comprising b7h3 chimeric antigen receptors

Abstract

To provide an improved therapeutic method targeting B7H3.SOLUTION: Provided is an isolated polynucleotide encoding a chimeric antigen receptor (CAR), comprising: a single-chain variable fragment (scFv), which specifically binds to B7H3; a transmembrane domain; a spacer located between the scFv and the transmembrane domain; and an intracellular signaling domain.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO PRIORITY AND RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 725,599, filed Aug. 31, 2018, which is expressly incorporated by reference in its entirety.

[0002] Electronic sequence listing This application has been filed via EFS-Web with an electronic Sequence Listing as an ASCII text file. The electronic Sequence Listing was created with the filename SCRI170WOSEQLIST.txt, was last updated on August 27, 2019, and is 15,402 bytes in size. The information set forth in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Embodiments of the methods and compositions provided herein relate to chimeric antigen receptors (CARs) that specifically bind to B7H3. Some embodiments relate to cell-based immunotherapy that targets tumors, such as tumors that contain B7H3+ cells. [Background technology]

[0004] B7 homolog 3 (B7H3), also known as CD276, is a type I transmembrane protein encoded by chromosome 15 in humans. Its extracellular domain contains two identical pairs of immunoglobulin variable and constant domains due to exon duplication in humans (four Ig-B7-H3 isoforms). The intracellular tail of B7H3 is short and has no known signaling motifs. B7H3 is ubiquitously expressed in a variety of species. Soluble forms cleaved from the cell surface by matrix metallopeptidases (MMPs) and generated by alternative splicing of introns are also detectable in human serum.

[0005] B7H3 is induced on antigen-presenting cells and plays a key role in suppressing T cell function. Importantly, B7H3 is highly overexpressed in a wide variety of human solid cancers and is often correlated with poor prognosis and poor clinical outcomes in patients. For example, many studies have reported that B7H3 is overexpressed in human malignancies, such as melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and other cancers. See, e.g., Wang J, et al., J Invest Dermatol. 133:2050-8; Hu Y, et al., Hematology. 20:187-95; Sun J, et al., OncoTargets Ther. 7:1979-86; Zang X, et al., Proc Natl Acad Sci US A. 104:19458-63; Zang X et al., Mod Pathol. 23:1104-12; Chen Y, et al., OncoTargets Ther. 7:1465-72; and Ingebrigtsen VA et al., BMC Cancer. 14:602. Immunochemical analysis has detected that 60%-93% of tumor tissues from patients with most types of cancer show aberrant expression of B7H3, whereas limited expression has been observed in normal healthy tissues. In most cases, high expression of B7H3 correlates with poor prognosis and clinical outcome, therefore, improved therapeutic approaches targeting B7H3 are needed. Summary of the Invention [Means for solving the problem]

[0006] Some embodiments of the methods and compositions provided herein include an isolated polynucleotide encoding a chimeric antigen receptor (CAR), comprising: A single-chain variable fragment (scFv) that specifically binds to B7H3; transmembrane domain; a spacer positioned between the scFv and the transmembrane domain; and Intracellular signaling domains The polynucleotide comprises:

[0007] In some embodiments, the scFv comprises a heavy chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:3. H ) CDR3, and a light chain variable region (V L ) CDR3.

[0008] In some embodiments, the scFv has a V sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1. H CDR1, V having at least 95% identity with the amino acid sequence of SEQ ID NO:2 H CDR2, V having at least 95% identity to the amino acid sequence of SEQ ID NO:4 L CDR1, and V having at least 95% identity with the amino acid sequence of SEQ ID NO:5 L Includes CDR2.

[0009] In some embodiments, the scFv has a V sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 13. H Includes the area.

[0010] In some embodiments, the scFv has a V sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 15. L Includes the area.

[0011] In some embodiments, the spacer comprises the amino acid sequence X1PPX2P.

[0012] In some embodiments, the spacer comprises a portion of a hinge region of a human antibody or an engineered variant thereof.

[0013] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer having at least 95% identity to the amino acid sequence of SEQ ID NO:18.

[0014] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer having at least 95% identity to the amino acid sequence of SEQ ID NO:19.

[0015] In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH2-CH3 spacer having at least 95% identity to the amino acid sequence of SEQ ID NO:20.

[0016] In some embodiments, the spacer comprises, consists of, or consists essentially of the IgG4 hinge-CH2(L235D, N297Q)-CH3 spacer.

[0017] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain (CD28tm) having at least 95% identity to the amino acid sequence of SEQ ID NO:30.

[0018] In some embodiments, the intracellular signaling domain comprises a first signaling domain and a costimulatory signaling domain, which may comprise all or a portion of CD3zeta in combination with a costimulatory signaling domain selected from the group consisting of the signaling domains of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, MyD88, and B7-H3, and combinations thereof.

[0019] In some embodiments, the intracellular signaling domain comprises a signaling portion of CD3ζ encoded by a nucleic acid having at least 95% identity to the nucleotide sequence of SEQ ID NO:31.

[0020] In some embodiments, the intracellular signaling domain comprises a signaling portion of 4-1BB encoded by a nucleic acid having at least 95% identity to the nucleotide sequence of SEQ ID NO:32.

[0021] Some embodiments further comprise a nucleic acid encoding an identification marker.

[0022] In some embodiments, the identifying marker is a truncated receptor. In some embodiments, the truncated receptor is selected from the group consisting of truncated EGFR (EGFRt), truncated HER2 (HER2t) and truncated CD19 (CD19t).

[0023] Some embodiments further comprise a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence comprises P2A or T2A.

[0024] Some embodiments further comprise a selection marker suitable for selecting cells comprising the isolated polynucleotide. In some embodiments, the selection marker comprises a transgene encoding a dihydrofolate reductase. In some embodiments, the transgene encoding the dihydrofolate reductase is a dihydrofolate reductase double mutant (DHFRdm). In some embodiments, the dihydrofolate reductase double mutant comprises a L22F amino acid mutation and a F31S amino acid mutation.

[0025] Some embodiments include a V having at least 95% identity to SEQ ID NO:9. H A nucleotide sequence encoding CDR3 and V having at least 95% identity with SEQ ID NO:12. L It further includes a nucleotide sequence encoding the CDR3.

[0026] Some embodiments include a V having at least 95% identity to SEQ ID NO:7. H A nucleotide sequence encoding CDR1, V having at least 95% identity to SEQ ID NO:8 H A nucleotide sequence encoding CDR2, V having at least 95% identity to SEQ ID NO:10 L A nucleotide sequence encoding CDR1 and V having at least 95% identity with SEQ ID NO:11. L It further includes a nucleotide sequence encoding CDR2.

[0027] Some embodiments comprise a heavy chain variable region (V) having at least 95% identity to SEQ ID NO:14. H )'s amino acid sequence.

[0028] Some embodiments comprise a light chain variable region (V) having at least 95% identity to SEQ ID NO: 16. L )'s amino acid sequence.

[0029] Some embodiments of the methods and compositions provided herein include a protein encoded by a polynucleotide according to any of the above embodiments associated with the isolated polynucleotide.

[0030] Some embodiments of the methods and compositions provided herein include a vector comprising an isolated polynucleotide according to any of the preceding embodiments relating to the isolated polynucleotide.

[0031] In some embodiments, the vector is a viral vector.

[0032] In some embodiments, the vector is a lentiviral vector or an adenoviral vector.

[0033] Some embodiments of the methods and compositions provided herein include a cell comprising an isolated polynucleotide according to any of the above embodiments associated with the isolated polynucleotide.

[0034] Some embodiments of the methods and compositions provided herein include a cell comprising a vector according to any of the above embodiments relating to said vector.

[0035] Some embodiments of the methods and compositions provided herein include a cell comprising a protein encoded by an isolated polynucleotide according to any of the above embodiments associated with the isolated polynucleotide.

[0036] In some embodiments, the cells are selected from the group consisting of CD8+ T cells and CD4+ T cells.

[0037] In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0038] In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells that contain CD45RA+, CD62L+, and CD4+, but lack CD45RO.

[0039] In some embodiments, the cells are progenitor T cells.

[0040] In some embodiments, the cells are hematopoietic stem cells.

[0041] Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising a cell according to any of the above embodiments relating to said cell and a pharma- ceutically acceptable excipient.

[0042] Some embodiments of the methods and compositions provided herein relate to a method of making a cell according to any of the above embodiments, comprising: introducing the isolated nucleic acid according to any of the preceding embodiments relating to said isolated nucleic acid into a lymphocyte; Culturing the lymphocytes containing the isolated nucleic acid in the presence of an agent selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and a cytokine; and Selectively enriching said lymphocytes containing said isolated nucleic acid. The present invention includes a method comprising the steps of:

[0043] In some embodiments, the selective enrichment step comprises contacting lymphocytes containing the isolated nucleic acid with a selection agent, hi some embodiments, the selection agent comprises methotrexate.

[0044] In some embodiments, the lymphocytes have a CD45RA-, CD45RO+ and CD62L+ phenotype.

[0045] In some embodiments, the lymphocytes are CD8+ lymphocytes or CD4+ lymphocytes.

[0046] In some embodiments, the cytokine is selected from the group consisting of IL-15, IL-7, IL-2 and IL-21.

[0047] Some embodiments further comprise the step of isolating the selectively enriched lymphocytes.

[0048] Some embodiments further comprise the step of contacting said selectively enriched lymphocytes with an antibody against a distinguishing marker expressed by said lymphocytes.

[0049] In some embodiments, the identifying marker comprises a truncated receptor. In some embodiments, the truncated receptor is selected from the group consisting of truncated EGFR (EGFRt), truncated HER2 (HER2t) and truncated CD19 (CD19t).

[0050] Some embodiments of the methods and compositions provided herein include a method of treating or alleviating a subject having a tumor comprising B7H3+ cells, comprising administering to the subject an effective amount of cells according to any of the embodiments relating to the cells.

[0051] In some embodiments, the cells are generated from cells derived from the subject.

[0052] In some embodiments, the administration is intravenous.

[0053] In some embodiments, the administration is locoregional administration at the site of a tumor in the subject.

[0054] In some embodiments, the tumor comprises a cancer selected from the group consisting of malignant melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, non-small cell lung cancer, bladder cancer, renal clear cell carcinoma, and glioma.

[0055] In some embodiments, the tumor comprises a glioma. In some embodiments, the tumor comprises a glioma selected from the group consisting of oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and intrinsic pontine glioma (DIPG). In some embodiments, the tumor comprises a glioblastoma multiforme (GBM).

[0056] Some embodiments further comprise administering an additional therapeutic regimen.

[0057] In some embodiments, the additional therapeutic regimen comprises radiation therapy.

[0058] In some embodiments, the additional therapeutic regimen comprises a chemotherapeutic compound, which may be 2'-deoxyazacytidine, 4-epidoxorubicin, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, Ara-C, BMS-214662, bortezomib, capecitabine, CHIR-258, cisplatin, cladribine, clofarabine, dacarbazine, decitabine, doxorubicin, etoposide, floxuridine, fludarabine, forodesine, FR01228, gefitinib, cefotaxime ... The agent is selected from the group consisting of mucitabine, hydroxyurea, KW-2449, lestautinib, lonafarnib, melphalan, midostaurin, mitomycin C, nelarabine, pentostatin, roscovitine, sapacitabine, semaxanib, sodium valproate, sorafenib, sunitinib, tanzutinib, teniposide, thiarabine, tipifarnib, trichostatin A, troxacitabine, vanadate, verapamil, vidazam, and zosuquidar trihydrochloride.

[0059] In some embodiments, administration of the cells and administration of the additional therapeutic regimen are sequential.

[0060] In some embodiments, administration of the cells and administration of the additional therapeutic regimen occur concurrently.

[0061] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0062] Some embodiments of the methods and compositions provided herein include a cell comprising a protein encoded by an isolated polynucleotide according to any of the foregoing embodiments relating to the isolated polynucleotide, for use in medicine.

[0063] Some embodiments of the methods and compositions provided herein include cells for use in treating a tumor that contains B7H3+ cells in a subject.

[0064] In some embodiments, the tumor comprises a cancer selected from the group consisting of malignant melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, non-small cell lung cancer, bladder cancer, renal clear cell carcinoma, and glioma. In some embodiments, the tumor comprises a glioma. In some embodiments, the tumor comprises a glioma selected from the group consisting of oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and intrinsic pontine glioma (DIPG). In some embodiments, the tumor comprises a glioblastoma multiforme (GBM).

[0065] In some embodiments, the cells are selected from the group consisting of CD8+ T cells and CD4+ T cells.

[0066] In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocytes are central memory T cells, and the central memory T cells are CD45RO+, CD62L+, and CD8+.

[0067] In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells that contain CD45RA+, CD62L+, and CD4+, but lack CD45RO.

[0068] In some embodiments, the cells are progenitor T cells.

[0069] In some embodiments, the cells are hematopoietic stem cells.

[0070] Some embodiments of the methods and compositions provided herein include an isolated polynucleotide comprising a heavy chain variable region (V) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3. H ) CDR3, and / or a light chain variable region (V L ) CDR3.

[0071] Some embodiments of the methods and compositions provided herein include an isolated polynucleotide comprising a heavy chain variable region (V) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1. H ) CDR1, a V having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2 H CDR2, a light chain variable region (V L ) CDR1, and / or a V having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:5 L It includes a polynucleotide encoding CDR2.

[0072] Some embodiments of the methods and compositions provided herein comprise a heavy chain variable region (V) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:13. H) is an isolated polynucleotide encoding the

[0073] Some embodiments of the methods and compositions provided herein comprise a light chain variable region (V) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:15. L ) is an isolated polynucleotide encoding the

[0074] Some embodiments of the methods and compositions provided herein include an isolated polypeptide or a composition comprising the isolated polypeptide, wherein the polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, or SEQ ID NO:15.

[0075] Some embodiments of the methods and compositions provided herein include a cell comprising an isolated polynucleotide according to any of the preceding embodiments relating to the isolated polynucleotide or a polypeptide according to any of the preceding embodiments relating to the polypeptide. [Brief description of the drawings]

[0076] [Figure 1A] Schematic diagram of polynucleotides encoding various B7H3 CARs.

[0077] [Figure 1B] Graph showing EGFRt discrimination of B7H3 CAR T cells containing CARs with short (S), medium (M) or long (L) spacer regions.

[0078] [Figure 1C] Photographs of Western blots showing CD3ζ expression in T cells transduced with polynucleotides encoding B7H3 CARs with a short (S) spacer region, a medium (M) spacer region, or a long (L) spacer region.

[0079] [Figure 1D] Figure 1 is a series of graphs showing the in vitro % specific lysis and cytokine release by B7H3 CAR T cells containing CARs with short (S), medium (M) or long (L) spacer regions when stimulated with Epd-110GH, PNET-109FH, D283 or Med411FH cell lines. The cytokines measured were IFNγ, IL-2 and B7H3 TNF.

[0080] [Figure 1E] A series of graphs showing the number of B7H3+U87 tumor cells, detected using the firefly luciferase (ffluc) marker, and survival rates examined by Kaplan-Meier curves in mice inoculated with U87 cells and treated with B7H3 CAR T cells containing CARs with short (S) or medium (M) spacer regions. (X) mock; (■) B7H3 CAR with short spacer region; (●) B7H3 CAR with medium spacer region.

[0081] [Figure 2A] 1 is a series of graphs showing expression of EGFRt, expression of immunoglobulin that binds protein L, and expression of Fc that binds anti-Fc in CD4+ B7H3 CAR T cells containing CARs with a short (S), medium (M) or long (L) spacer region.

[0082] [Figure 2B] Photograph of a Western blot showing expression of CD3ζ in various clones of CD4+ or CD8+ cells transduced with polynucleotides encoding B7H3 CAR with a short (S), medium (M) or long (L) spacer region.

[0083] [Figure 2C]A series of graphs showing the percentage of specific lysis in vitro by B7H3 CAR T cells containing CARs with a short (S), medium (M) or long (L) spacer region when stimulated with U87 cells, modified U87 cells lacking expression of B7H3, Be2 cells or D283 cells: (X) mock; (▲) B7H3 CAR with a short spacer region; (○) B7H3 CAR with a medium spacer region; and (■) B7H3 CAR with a long spacer region.

[0084] [Figure 2D] 1 is a series of graphs showing cytokine release by CD4+ B7H3 CAR T cells or CD8+ B7H3 CAR T cells containing CARs with short (S), medium (M) or long (L) spacer regions upon stimulation with Jurkat, Jurkat OKT3, U87, modified U87 cells lacking B7H3 expression, Be2 or D283 cells. Cytokines measured were IFNγ, IL-2 and TNF.

[0085] [Figure 2E] A series of graphs showing the number of B7H3+U87 tumor cells detected using the ffluc marker and survival examined by Kaplan-Meier curves in mice inoculated with U87 cells and treated with CD8+ B7H3 CAR T cells containing CARs with a short (S), medium (M) or long (L) spacer region: (X) mock; (●) B7H3 CAR with a short spacer region; (●) B7H3 CAR with a medium spacer region; and (▲) B7H3 CAR with a long spacer region.

[0086] [Figure 3A]1 is a series of graphs showing expression of EGFRt, human Fab and Fc (which binds anti-Fc) in CD4+ cells transduced with a polynucleotide encoding B7H3 CAR with a medium length (M) spacer region and a DHFRdm selection marker.

[0087] [Figure 3B] Figure 1 is a series of graphs showing cytokine release by CD4+ B7H3 CAR T cells stimulated with Jurkat cells, Jurkat OKT3 cells, U87 cells, modified U87 cells lacking B7H3 expression, Be2 cells, and D283 cells. The cytokines measured were IFNγ, IL-2, and TNF.

[0088] [Figure 4] 1 is a series of graphs showing data regarding lysis of B7H3 positive target cell lines by T cells (Example 4).

[0089] [Diagram 5] 1 is a series of graphs showing data regarding cytokine production by T cells co-cultured with B7H3 positive target cell lines (Example 5).

[0090] [Figure 6] 1 is a series of graphs showing data regarding eradication of U87 tumor cells by T cells in vivo (Example 6).

[0091] [Figure 7] 1 is a series of graphs showing data regarding eradication of subcutaneous Be2 neuroblastoma cells by T cells in vivo (Example 7).

[0092] [Figure 8] 1 is a series of graphs showing data regarding eradication of subcutaneous Be2 neuroblastoma cells by T cells in vivo (Example 8).

[0093] [Figure 9]FIG. 1 is a series of graphs showing data on eradication of U251T glioma cells by CD8+ B7H3 CAR T cells in vivo (Example 9).

[0094] [Figure 10] FIG. 1 is a series of graphs showing data on the eradication of diffuse intrinsic pontine glioma (DIPG) primary tumor cells by CD8+ B7H3 CAR T cells in vivo (Example 10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Definition of Terms As used herein, the term "nucleic acid" or "nucleic acid molecule" has its common and ordinary meaning in the context of the present specification, including, but not limited to, polynucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and / or fragments obtained by ligation, cleavage, endonuclease action and / or exonuclease action. Nucleic acid molecules may be composed of natural nucleotide monomers (such as DNA and RNA), analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety and / or in the pyrimidine or purine base moiety. Modifications in the sugar moiety include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines and / or azide groups, and the sugar moiety may be etherified or esterified. Additionally, the entire sugar moiety may be replaced with conformationally and electronically similar structures, such as, for example, azasugars and / or carbocyclic sugar analogs. Modified base moieties include alkylated purines and / or pyrimidines, acylated purines or pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Linkages similar to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoroamidate, and the like. "Nucleic acid molecule" also includes so-called "peptide nucleic acids," which include natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. In some embodiments, a nucleic acid sequence is provided that encodes a protein. In some embodiments, the nucleic acid is RNA or DNA.

[0096] As used herein, the term "antibody" has its general and usual meaning in the context of this specification, including, but not limited to, a large Y-shaped protein produced by plasma cells that has the function of identifying and neutralizing foreign substances such as bacteria and viruses in the immune system. In some contexts, "antibody" refers to an antigen-binding fragment of an antibody. An antibody protein may contain four polypeptide chains, i.e., two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain is composed of structural domains called immunoglobulin domains. These domains can contain 70 to 110 amino acids and are classified into various categories based on their size and function. A chimeric antigen receptor can include a ligand-binding domain that includes an antibody fragment (preferably an antigen-binding fragment). In some embodiments, a nucleic acid encoding a chimeric antigen receptor (CAR), a) a first polynucleotide encoding a ligand-binding domain that binds to and / or interacts with a human B7H3 protein; b) a second polynucleotide encoding a polypeptide spacer of sufficient length to allow said ligand binding domain to interact with said human B7H3 protein; c) a third polynucleotide encoding a transmembrane domain, and d) a fourth polynucleotide encoding an intracellular signaling domain. The present invention provides a nucleic acid comprising: In some embodiments, the ligand binding domain is an antibody fragment. Examples of antibodies or binding fragments thereof capable of binding to a target moiety include monoclonal antibodies, bispecific antibodies, Fab, Fab2, Fab3, scFv, Bis-scFv, minibodies, triabodies, diabodies, tetrabodies, VhH domains, V-NAR domains, IgNAR, and camelid Ig. Further examples of antibodies include IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgE, IgD, and IgA. Further examples of antibodies include human antibodies, humanized antibodies, and chimeric antibodies.

[0097] As used herein, the term "single chain variable region fragment" or "scFv" has its common, ordinary meaning in the context of this specification, and includes, for example, a heavy chain variable region (V) of an immunoglobulin linked by a short linker peptide. H ) and the light chain variable region (V L In some embodiments, the linker may include glycine for flexibility and hydrophilic amino acids (e.g., serine or threonine) for solubility. The linker may include, but is not limited to, a fusion protein comprising a V H N-terminus and V L Alternatively, the C-terminus of V H The C-terminus of L The N-terminus of the CAR may also be linked. In some embodiments, the ligand binding domain present on the CAR is a single chain variable fragment (scFv). In some embodiments, the scFv domain present on the CAR is specific for the B7H3 protein.

[0098] As used herein, "vector", "expression vector" or "construct" has its general and ordinary meaning in the context of the present specification, including, but not limited to, a nucleic acid used to introduce a heterologous nucleic acid into a cell, which contains various regulatory elements and thus allows the expression of the heterologous nucleic acid in the cell. Vectors include, but are not limited to, a plasmid, a minicircle, a yeast and / or a viral genome. In some embodiments, the vector is a plasmid, a minicircle or a viral genome. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is a lentivirus vector. In some embodiments, the vector is a foamy virus vector, an adenovirus vector, a retrovirus vector, or a lentivirus vector.

[0099] As used herein, the term "chimeric antigen receptor", "CAR" or "chimeric T cell receptor" has its general and ordinary meaning in the context of the present specification, including but not limited to a synthetically designed receptor that includes a ligand binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder, and the ligand binding domain is linked to one or more intracellular signaling domains (e.g., costimulatory domains) of a T cell receptor or other receptor via a spacer domain. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs). CARs are genetically engineered receptors that can be grafted with any specificity into cells that express immune receptors. Some researchers understand "chimeric antigen receptors" or "CARs" to include an antibody or antibody fragment, a spacer, a signaling domain, and a transmembrane region. However, the CARs described herein have been modified in various components or domains, such as ligand binding regions (e.g., antibody fragments, scFvs, or portions thereof), spacers, transmembrane domains, and / or signaling domains, with surprising effects, and therefore the components of the CAR are often distinguished as separate elements throughout the disclosure. In some embodiments, the spacer of the chimeric antigen receptor is selected (e.g., the spacer is of a particular amino acid length) to confer desired binding properties to the CAR. CARs with spacer lengths of various lengths are then screened, e.g., on cells, for their ability to bind or interact with the target moiety to which the CAR is directed.

[0100] As used herein, a "T cell" or "T lymphocyte" may be a T cell obtained from any type of mammal, including monkeys, dogs, humans, etc., but is preferably a T cell obtained from a primate. In some embodiments, the T cells are allogeneic (same species but from a different donor) to the recipient subject to whom the T cells will be administered, e.g., in the form of a therapeutic composition, or to the recipient subject to whom the T cells have been administered. In some embodiments, the T cells are autologous (donor and recipient are the same). In some embodiments, the T cells are syngeneic (donor and recipient are different but identical twins).

[0101] Herein, mature T cells express CD4 as a surface protein and are referred to as CD4+ T cells. CD4+ T cells are generally treated as being destined to play a role as helper T cells in the immune system. For example, when an antigen-presenting cell expresses an antigen on class II MHC, CD4+ cells help the antigen-presenting cell through a combination of cell-cell interactions (e.g., CD40 and / or CD40L) and cytokines. However, there are rare exceptions, e.g., regulatory T cells, natural killer cells, and subgroups of cytotoxic T cells also express CD4. None of these CD4+ expressing T cell populations are considered to be helper T cells.

[0102] As used herein, "central memory" T cells (or "T CMThe term "cell-specific antigen-specific antigen" has its common and ordinary meaning in the context of this specification, including, but not limited to, antigen-experienced cytotoxic T lymphocytes (CTLs) that express CD62L or CCR-7 and / or CD45RO on their surface, but do not express CD45RA or have reduced expression of CD45RA, as compared to naive cells. In some embodiments, central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO and / or CD95, and have reduced expression of CD54RA, as compared to naive cells.

[0103] As used herein, "effector" T cells or "T E " in the context of this specification has its general and ordinary meaning, and includes, for example, but is not limited to, antigen-experienced cytotoxic T lymphocytes that do not express CD62L, CCR7 and CD28 or have reduced expression of CD62L, CCR7 and CD28, and / or are positive for granzyme B and / or perforin, compared to central memory T cells or naive T cells.

[0104] As used herein, the term "progenitor T cell" has its general and ordinary meaning in the context of this specification, including, but not limited to, lymphoid progenitor cells that can migrate to the thymus and become progenitor T cells and do not express T cell receptors. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (lymphoid progenitor cells) derived from hematopoietic stem cells settle in the thymus and expand by cell division, generating a large population of immature thymocytes. Very early thymocytes express neither CD4 nor CD8, and are therefore double negative (CD4 - CD8 - ) cells. As they develop, they are classified as double positive thymocytes (CD4 + CD8 + ) and finally became single positive (CD4 + CD8 - or CD4 -CD8 + ) They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0105] As used herein, the term "pharmaceutical excipient" or "pharmaceutical carrier" has its common and ordinary meaning in the context of the present specification, including, but not limited to, a carrier or inert vehicle used as a vehicle for formulating and / or administering a pharma- ceutical active agent or T cells for therapeutic purposes. Carriers include polymeric micelles, liposomes, lipoprotein-based carriers, nanoparticle carriers, dendrimers, and / or other carriers for T cells known to those skilled in the art. Ideal carriers or excipients include those that are non-toxic, biocompatible, non-immunogenic, biodegradable, and not recognized by the host's defense mechanisms.

[0106] Detailed Description Some embodiments of the methods and compositions provided herein relate to chimeric antigen receptors (CARs) that specifically bind to human B7 homolog 3 (B7H3). Some embodiments relate to polynucleotides encoding CARs that specifically bind to B7H3, cells expressing such CARs, and using such cells to treat subjects with B7H3+ tumors.

[0107] Some of the embodiments provided herein relate to B7H3 CAR and polynucleotides encoding such CAR. Some of the embodiments of B7H3 CAR provided herein have particularly significant activity in vitro and in vivo. For example, certain embodiments of B7H3 CAR expressed in T cells have been demonstrated to have significantly increased activity in stimulating cytokine release in vitro, significantly increased activity in suppressing tumor cell proliferation in vivo, and significantly prolong the survival of subjects. Furthermore, certain embodiments of B7H3 CAR expressed in T cells have been demonstrated to have significant activity against several types of central nervous system tumor cells in vitro, and significant activity in an in vivo xenograft model with human glioblastoma cells.

[0108] In some embodiments, B7H3 CAR T cells can be generated by transducing a polynucleotide encoding a B7H3 CAR into T cells. The transduced cells can express the B7H3 CAR and target cells expressing the B7H3 antigen. In some embodiments, T cells can be harvested from a subject. The harvested T cells can be engineered ex vivo to express the B7H3 CAR and reintroduced into the subject. The reintroduced T cells can target tumor cells expressing the B7H3 antigen in the subject. Some embodiments of the B7H3 CAR provided herein include a modified IgG4 hinge region that can enhance the stability and activity of the CAR. In some embodiments, the B7H3 CAR includes a CD28 transmembrane region and provides effector function through a combination of intracellular costimulatory molecules. In some embodiments, the polynucleotide encoding the B7H3 CAR can encode a second polypeptide, such as an identification marker, such as a truncated EGFR (EGFRt). Such identification markers may be useful for isolating cells that co-express the identification markers, and may be useful for measuring the amount of cells expressing the identification markers in subjects treated with such cells. As described herein, the B7H3 CAR has been successfully expressed in CD4 T cells and CD8 T cells, and has been confirmed to confer potent and specific anti-tumor activity using a series of in vitro and in vivo assays.

[0109] When using scFv as a targeting moiety, the design of CAR becomes more complicated because the scFv is related to the recognition and target by T cells. While the classical interaction between TCR and the HLA molecule it recognizes depends on spatial relationship, the CAR and its target discrimination ability depend on multiple parameters such as the location of the epitope, the structure of the target, and the length and flexibility of the extracellular spacer of the CAR. The exact mechanism underlying the effect of different lengths of extracellular spacer on the binding of CAR to the target is still unclear. In an embodiment of the present disclosure, by incorporating different lengths of IgG4 heavy chain spacer into B7H3 CAR, it is found that a certain B7H3 CAR has remarkable and unexpected activity in vitro and in vivo.

[0110] Chimeric antigen receptors (CARs) Some embodiments of the methods and compositions provided herein include B7H3 CAR and isolated polynucleotides encoding the B7H3 CAR. As used herein, the term "B7H3 CAR" refers to a CAR that targets the B7H3 antigen. In some embodiments, the CAR may comprise a synthetically engineered protein that includes an antigen-binding domain that specifically binds to a target, a spacer domain that links the antigen-binding domain to a transmembrane domain, and an intracellular signaling domain. In some embodiments, the antigen-binding domain may comprise an antibody, an antigen-binding fragment of an antibody, or a fusion protein derived from an antibody, such as a single chain variable fragment (scFv). An scFv may comprise a single chain Fv antibody, which is a single polypeptide chain formed by linking the variable domains of the heavy and light chains of a conventional two-chain antibody. In some embodiments, the intracellular signaling domain may comprise a signaling domain and a costimulatory domain.

[0111] Some of the embodiments of CARs provided herein include an antigen binding domain that specifically binds to the B7H3 antigen. In some embodiments, the antigen binding domain of the CAR may be derived from an antibody that specifically binds to B7H3. Examples of such antibodies are disclosed in International Patent Publication WO 2011109400, US Patent Publication No. 2013 / 0149236, and Loo D., et al., (2012) Clin Cancer Res; 18(14); 3834-45, which are expressly incorporated herein by reference in their entireties. The antibody may include a heavy chain variable region (VCL) that may include complementarity determining regions (CDRs), such as complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3). H The antibody may also include a light chain variable region (V) that may include complementarity determining regions such as complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3). L ) may also be included.

[0112] In some embodiments, the antigen binding domain of a CAR that specifically binds to B7H3 is a V H CDR3, V derived from an antibody that specifically binds B7H3 H CDR1, and / or V from an antibody that specifically binds to B7H3 H In some embodiments, the antigen-binding domain of a CAR that specifically binds to B7H3 comprises at least CDR2. H In some embodiments, the antigen binding domain of a CAR that specifically binds to B7H3 may comprise at least a V region derived from an antibody that specifically binds to B7H3. L CDR3, V derived from an antibody that specifically binds B7H3 L CDR1, and / or V from an antibody that specifically binds to B7H3 LIn some embodiments, the antigen-binding domain of a CAR that specifically binds to B7H3 comprises at least CDR2. L The region may include at least

[0113] In some embodiments, the antigen binding domain of a CAR that specifically binds to B7H3 comprises an scFv derived from an antibody that specifically binds to B7H3. In some embodiments, the scFv comprises a V H CDR3, V H CDR1 and / or V H In some embodiments, the scFv comprises a V CDR2 derived from an antibody that specifically binds to B7H3. H In some embodiments, the scFv comprises a V region derived from an antibody that specifically binds B7H3. L CDR3, V L CDR1 and / or V L In some embodiments, the scFv comprises a V CDR2 derived from an antibody that specifically binds to B7H3. L In some embodiments, the scFv comprises a V region derived from an antibody that specifically binds B7H3. H Area and V L or such a V H Area and V L Contains the CDR elements of the V H Area and V L The domains can be linked to each other via a linker. H Area and V L The area is V H -V L or V H -V L They can be linked in the following order.

[0114] Exemplary amino acid and nucleotide sequences useful in embodiments of the methods and compositions provided herein are provided in Table 1. [Table 1] TIFF2025066785000002.tif68165

[0115] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1. H CDR1 may be included, and V consisting of this amino acid sequence H In some embodiments, the V H CDR1 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:7.

[0116] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2. H CDR2 may be included, and V consisting of this amino acid sequence H In some embodiments, the V H CDR2 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:8.

[0117] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:3. H CDR3 may be included, and V consisting of this amino acid sequence H In some embodiments, the V HThe CDR3 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:9.

[0118] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 13. H A V region may be included, which comprises this amino acid sequence. H In some embodiments, V H The region may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:14.

[0119] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:4. L CDR1 may be included, and V consisting of this amino acid sequence L In some embodiments, the V L CDR1 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:10.

[0120] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:5. L CDR2 may be included, and V consisting of this amino acid sequence L In some embodiments, the V LCDR2 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:11.

[0121] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:6. L CDR3 may be included, and V consisting of this amino acid sequence L In some embodiments, the V L The CDR3 may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:12.

[0122] In some embodiments, a CAR that specifically binds to B7H3 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 15. L A V region may be included, which comprises this amino acid sequence. L In some embodiments, V L The region may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:16.

[0123] In some embodiments, the percentage of identity between two sequences, such as between two amino acid sequences or between two nucleotide sequences, can be determined, for example, by aligning the sequences for the purpose of performing an optimized comparison, for example, by inserting gaps in the sequence of the first sequence to align the sequences. Then, the amino acids or nucleotides at corresponding positions are compared, and the percentage of identity between the two sequences is determined as a function of the number of positions where the amino acids or nucleotides match between the two sequences. For example, the percentage of identity (%) is calculated as (number of matching positions / total number of positions)×100. The comparison between the two sequences can be actually performed by a known method using, for example, a mathematical algorithm. Specific examples of such mathematical algorithms include, but are not limited to, those described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al., Nucleic Acids Res., 29:2994-3005 (2001), which is incorporated herein by reference in its entirety. In some embodiments of the methods and compositions provided herein, the nucleic acid sequence of a polynucleotide encoding a CAR or a component thereof may consist of, consist substantially of, or include a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a nucleotide sequence provided herein.In some embodiments of the methods and compositions provided herein, the amino acid sequence encoding a CAR polypeptide or a component thereof may consist of, consist substantially of, or comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence provided herein.

[0124] In some embodiments, the amino acid sequence may include conservative amino acid substitutions. Conservative amino acid changes result in synonymous changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids with similar polarity have similar functions, resulting in synonymous changes in the amino acid sequence of the resulting peptide. Substitutions that do not change the charge by replacing a particular residue with a smaller residue may also be considered conservative substitutions, even if these residues belong to different groups (e.g., replacing phenylalanine with the smaller isoleucine). In some embodiments, conservative amino acid substitutions may include the use of amino acid analogs or variants. An example of a family of conservative amino acid substitutions is listed in Table 2. [Table 2]

[0125] In some embodiments, the B7H3 CAR may include a spacer domain that links the antigen-binding domain to the transmembrane domain. In some embodiments, the use of a spacer domain of appropriate length can provide flexibility to the antigen-binding domain, thereby optimizing binding to the target antigen. Examples of spacer domains useful in some of the embodiments of the methods and compositions provided herein are described in International Patent Publication WO 2014031687, US Patent Publication No. 20180200298, and Kunkele A., et al. (2015) Cancer Immunol Res; 3(4); 368-79, which are incorporated herein by reference in their entirety.

[0126] In some embodiments, the spacer domain may be derived from at least a portion of the hinge region of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the spacer domain may be derived from the CH2 and / or CH3 regions of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the spacer domain may comprise an amino acid sequence of an upper hinge region located between the heavy chain variable region and the core region, and an amino acid sequence of the core hinge region including a polyproline region. In some embodiments, the upper hinge region has 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acids. In some embodiments, the spacer region comprises the amino acid sequence X1PPX2P (SEQ ID NO: 17). In some embodiments, X1 is cysteine, glycine, or arginine, and X2 is cysteine ​​or threonine. In some embodiments, the spacer domain may comprise a modified hinge region of an IgG, for example the modified hinge region may have at least 90%, 92%, 95% or 100% sequence identity to the hinge region of an IgG, or may have sequence identity within a range defined by any two of these percentages.

[0127] In some embodiments, the length of the spacer region is at least or at least about 10 to 229 amino acids long, at least or at least about 10 to 200 amino acids long, at least or at least about 10 to 175 amino acids long, at least or at least about 10 to 150 amino acids long, at least or at least about 10 to 125 amino acids long, at least or at least about 10 to 100 amino acids long, at least or at least about 10 to 100 amino acids long, It may be 10 to 75 amino acids or at least about 10 to 75 amino acids long, at least 10 to 50 amino acids or at least about 10 to 50 amino acids long, at least 10 to 40 amino acids or at least about 10 to 40 amino acids long, at least 10 to 30 amino acids or at least about 10 to 30 amino acids long, at least 10 to 20 amino acids or at least about 10 to 20 amino acids long, or at least 10 to 15 amino acids long, or may be a length indicated by an integer between the upper and lower limits of any of the ranges listed here. In some embodiments, the spacer region is (at least 1 amino acid long and) 12 amino acids or less or (at least 1 amino acid long and) 119 amino acids or less or (at least 1 amino acid long and) 229 amino acids or less or (at least 1 amino acid long and) 229 amino acids or less.

[0128] In some embodiments, the spacer region may comprise, consist of, or consist essentially of a human IgG4 hinge spacer. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a polypeptide having the amino acid sequence of SEQ ID NO: 18. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a human IgG4 hinge-CH3 spacer. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a polypeptide having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a human IgG4 hinge-CH3-CH4 spacer. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a polypeptide having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the spacer region may comprise, consist of, or consist essentially of a human IgG4 hinge-CH2(L235D, N297Q)-CHE spacer. An example of an amino acid sequence of a spacer domain useful in embodiments of the methods and compositions provided herein is listed in Table 3. In some embodiments, the spacer region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the amino acid sequences listed in Table 3. [Table 3]

[0129] In some embodiments, the B7H3 CAR may comprise a transmembrane domain. The transmembrane domain may serve to anchor the CAR to the cell membrane. In some embodiments, the transmembrane domain may be derived from a membrane-associated protein or a transmembrane protein. In some embodiments, the length of the transmembrane domain may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids, or may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids in length. In some embodiments, the transmembrane domain may comprise the transmembrane region of the α, β or ζ chain of the T cell receptor, e.g., CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. In some embodiments, the transmembrane domain may be derived from the transmembrane domain of CD28 (CD28tm). An example of an amino acid sequence and a nucleotide sequence of a CD28tm domain useful in embodiments of the methods and compositions provided herein is provided in Table 4. In some embodiments, the transmembrane domain may be encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:29. [Table 4]

[0130] In some embodiments, the B7H3 CAR may comprise an intracellular signaling domain linked to a transmembrane domain. The intracellular signaling domain may activate a function of a cell when the antigen binding domain binds to a target antigen. In some embodiments, the intracellular signaling domain may activate a function of a CAR-expressing cell, such as a T cell expressing the CAR. In some embodiments, the intracellular signaling domain comprises one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain may comprise a domain derived from at least a portion of an intracellular signaling domain that activates at least one function of a transduced cell, such as a T cell. Activation of T cells occurs via at least two intracellular signaling proteins, one of which is an intracellular signaling protein (e.g., a first cytoplasmic signaling protein) that initiates antigen-dependent primary activation and provides a T cell receptor-like signal, and the other is an intracellular signaling protein (e.g., a second cytoplasmic signaling protein) that acts in an antigen-independent manner to provide a second signal, i.e., a costimulatory signal. In some embodiments, the intracellular signaling domain may comprise a functional domain of a first cytoplasmic signaling protein. In some embodiments, the intracellular signaling domain may comprise a functional domain of a first cytoplasmic signaling protein and at least one functional domain of one or more second cytoplasmic signaling proteins.

[0131] In some embodiments, the signaling domain, such as the first signaling domain or the costimulatory signaling domain, includes an intracellular or cytoplasmic domain of a protein or receptor protein that can interact with a component within the cell to relay or participate in relaying a signal. In some embodiments, such interaction occurs by signaling by the intracellular domain through specific protein-protein or protein-ligand interactions with an effector molecule or protein, which can then initiate a chain reaction of signaling to send the signal to its destination. In some embodiments, the signaling domain includes a costimulatory domain. In some aspects, the costimulatory domain includes a signaling moiety that provides a signal to the T cell that enhances a response, such as an immune response, activation, proliferation, differentiation, cytokine secretion, cytolytic activity, perforin activity, and / or granzyme activity, in addition to a first signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex, such as a T cell effector response. In some embodiments, the intracellular signaling domain and / or costimulatory domain can include all or a portion of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3, and / or all or a portion of a ligand that specifically binds CD83.

[0132] The first intracellular signaling protein that acts in a stimulatory manner may contain a signaling motif known as an intracellular receptor tyrosine-based activation motif (ITAM). First intracellular signaling domains that contain ITAMs include those derived from CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, or CD66d.

[0133] In some embodiments, the intracellular signaling domain may comprise all or a portion of the signaling domain of CD3zeta or a variant thereof and all or a portion of the signaling domain of 4-1BB or a variant thereof. An example of a nucleotide sequence encoding the signaling domain of CD3zeta and a nucleotide sequence encoding the signaling domain of 4-1BB is provided in Table 5. In some embodiments, the intracellular signaling domain may comprise an amino acid sequence encoded by a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the amino acid sequences provided in Table 5. [Table 5]

[0134] In some embodiments, the polynucleotide encoding the B7H3 CAR may be multicistronic and may encode two or more polypeptides. In some embodiments, the polynucleotide encoding the CAR may contain elements that allow translation of multiple genes from a single polynucleotide. In some embodiments, the elements may include an internal ribosome entry site (IRES) or a ribosome skipping sequence. A "ribosome skipping sequence" can include a sequence that functions to cause a translating ribosome to "skip" the ribosome skipping sequence and resume translation from the region immediately following the ribosome skipping sequence without forming a peptide bond. For example, some viruses have ribosome skipping sequences that allow multiple proteins to be translated consecutively from a single nucleic acid, and the translated proteins are obtained as separate proteins without being linked by peptide bonds. In some embodiments, the ribosome skipping sequence is a P2A sequence, a T2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the ribosome skipping sequence is a T2A sequence. An example of a nucleotide sequence of T2A useful in embodiments of the methods and compositions provided herein is provided in Table 5.

[0135] In some embodiments, the polynucleotide encoding the CAR may have an internal ribosome entry site (IRES) or ribosome skip sequence between the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the second polypeptide. In some embodiments, the CAR and the second polypeptide may be co-expressed in a cell. In some embodiments, the second polypeptide may encode an identification marker and / or a selection marker, such as a protein that can be used to identify, isolate, and / or enrich cells expressing the identification marker and / or selection marker. Examples of such markers include truncated receptors that can be used to identify expressing cells but have minimal activity. Examples of truncated receptors include truncated epidermal growth factor receptor (EGFRt), as described in U.S. Pat. No. 8,802,374, which is incorporated herein by reference in its entirety. Table 5 provides an example of a nucleotide sequence encoding an EGFRt that is useful in embodiments of the methods and compositions provided herein. Further examples of truncated receptors include truncated HER2 (HER2t) and truncated CD19 (CD19t). Examples of selection markers include a transgene encoding dihydrofolate reductase. In some embodiments, the dihydrofolate reductase may be a dihydrofolate reductase double mutant (DHFRdm). In some such embodiments, the dihydrofolate reductase double mutant comprises an L22F amino acid mutation and an F31S amino acid mutation. Further examples of selection markers include the hygromycin B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which codes for resistance to the antibiotic G418, and the adenosine deaminase gene (ADA). In some embodiments, a differentiation marker such as EGFRt can be used to isolate cell populations.In some embodiments, an antibody against the identification marker can be used to capture cells expressing the identification marker, such as EGFRt. The antibody can include a first binding partner, such as biotin or streptavidin. The antibody can be captured using a second binding partner, such as biotin or streptavidin, attached to a substrate, such as a bead, such as a magnetic bead. In some embodiments, the amount of CAR T cells in a subject to which the CAR T cells have been administered can be measured using an identification marker, such as EGFRt.

[0136] Some of the embodiments of the methods and compositions provided herein may include a vector comprising an isolated polynucleotide encoding a CAR that specifically binds to B7H3. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector or an adenoviral vector.

[0137] How CAR T cells are made Some embodiments of the methods and compositions provided herein may include methods of making cells comprising a polynucleotide encoding a B7H3 CAR, and compositions comprising such cells. Some of these embodiments include, for example, introducing an isolated polynucleotide encoding a B7H3 CAR into lymphocytes; culturing the lymphocytes comprising the isolated polynucleotide in the presence of an agent that promotes the expansion and proliferation of a cell population comprising the isolated polynucleotide, such as an agent selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and a cytokine; and selectively enriching the lymphocytes comprising the isolated polynucleotide. In some embodiments, the selective enrichment includes contacting the lymphocytes comprising the isolated nucleic acid with a selection reagent. In some embodiments, the selection reagent comprises methotrexate. In some embodiments, the lymphocytes have a phenotype of CD45RA-, CD45RO+, and CD62L+. In some embodiments, the lymphocytes are CD8+ lymphocytes or CD4+ lymphocytes. In some embodiments, the cytokine is selected from the group consisting of IL-15, IL-7, IL-2 and IL-21. Some embodiments further comprise isolating the selectively enriched lymphocytes. In some embodiments, the method comprises contacting the selectively enriched lymphocytes with an antibody against a distinguishing marker expressed by the lymphocytes. In some embodiments, the distinguishing marker comprises a truncated receptor. In some embodiments, the truncated receptor is selected from the group consisting of truncated EGFR (EGFRt), truncated HER2 (HER2t) and truncated CD19 (CD19t). One example of such a method is described in U.S. Patent Publication No. 20180200298, which is incorporated herein by reference in its entirety.

[0138] Some embodiments of the methods and compositions provided herein may include the selection and sorting of T lymphocyte populations and the modification of T lymphocyte populations. In some embodiments involving the selection and sorting of T lymphocyte populations, the T lymphocytes may be collected by known techniques and enriched or depleted by known techniques, such as methods that utilize affinity binding to antibodies, such as flow cytometry and / or immunomagnetic selection. After the enrichment and / or depletion steps, the desired T lymphocytes may be expanded in vitro by known techniques. Such known expansion methods include, for example, those described in U.S. Patent No. 6,040,177, which is incorporated herein by reference in its entirety. In some embodiments, the T cells are autologous T cells obtained from the patient.

[0139] In some embodiments, a desired T cell population or T cell subpopulation may be expanded in vitro by adding a pre-cultured T lymphocyte population to culture medium and then adding feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMCs)) to the culture medium. The non-dividing feeder cells may comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are gamma-irradiated. In some embodiments, the expansion method may further comprise adding EBV-transformed non-dividing lymphoblastoid cells (LCLs) as feeder cells. The LCLs may be gamma-irradiated. In some embodiments, the expansion method may comprise adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium. In some embodiments, the expansion method may comprise adding IL-2 and / or IL-15 to the culture medium (e.g., the concentration of IL-2 is at least about 10 units / ml). In some embodiments, the expanded T lymphocytes include CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T lymphocytes, which may be specific for antigens present in human tumors or pathogens. Before or after expansion, the initially isolated cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive, memory and effector T cell subpopulations, respectively.

[0140] CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive CD8+ cells, central memory CD8+ cells, and effector memory CD8+ cells by identifying cell surface antigens associated with each of these CD8+ cells. In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets from CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, central memory T cells (T CMExpression of phenotypic markers of T cells include CD45RO, CD62L, CCR7, CD28, CD3 and / or CD127, and / or Granzyme B is negative or shows low expression. In some embodiments, central memory T cells are CD45RO+, CD62L+ and / or CD8+ T cells. In some embodiments, effector T cells (T E ) are negative for CD62L, CCR7, CD28 and / or CD127, and / or positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127 and / or CD45RA.

[0141] In some embodiments, the presence or absence of a particular cell surface marker on the surface of a cell population can be confirmed by flow cytometry using staining with an antibody specific for the surface marker and an isotype-matched control antibody. A cell population is negative for a particular marker when it is stained with an antibody that stains more intensely with the specific antibody than with an isotype control, whereas a cell population is positive when it stains more uniformly with the specific antibody than with an isotype control. In some embodiments, a reduction in expression of one or more markers is measured by a 1 log reduction in mean fluorescence intensity compared to a control cell population. 10and / or a decrease in the percentage of cells expressing one or more markers to at least 20% of the total cells, at least 25% of the total cells, at least 30% of the total cells, at least 35% of the total cells, at least 40% of the total cells, at least 45% of the total cells, at least 50% of the total cells, at least 55% of the total cells, at least 60% of the total cells, at least 65% of the total cells, at least 70% of the total cells, at least 75% of the total cells, at least 80% of the total cells, at least 85% of the total cells, at least 90% of the total cells, at least 95% of the total cells, or at least 100% of the total cells. This refers to a decrease of at least about 20%, at least about 25% of the total cells, at least about 30% of the total cells, at least about 35% of the total cells, at least about 40% of the total cells, at least about 45% of the total cells, at least about 50% of the total cells, at least about 55% of the total cells, at least about 60% of the total cells, at least about 65% of the total cells, at least about 70% of the total cells, at least about 75% of the total cells, at least about 80% of the total cells, at least about 85% of the total cells, at least about 90% of the total cells, at least about 95% of the total cells, or at least about 100% of the total cells, or any percentage in the range of 20-100%.In some embodiments, a cell population being positive for one or more markers refers to the percentage of cells expressing one or more markers relative to a control cell population that is at least 50% of the total cells, at least 55% of the total cells, at least 60% of the total cells, at least 65% of the total cells, at least 70% of the total cells, at least 75% of the total cells, at least 80% of the total cells, at least 85% of the total cells, at least 90% of the total cells, at least 95% of the total cells, or at least 100% of the total cells, or at least about 50% of the total cells, at least about 55% of the total cells, at least about 60% of the total cells, at least about 65% of the total cells, at least about 70% of the total cells, at least about 75% of the total cells, at least about 80% of the total cells, at least about 85% of the total cells, at least about 90% of the total cells, at least about 95% of the total cells, or at least about 100% of the total cells, or any percentage in between.

[0142] CD4+ helper T cells are sorted into naive cells, central memory cells and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ or CD45RO+. In some embodiments, effector CD4+ cells are CD62L- or CD45RO-.

[0143] In some embodiments, antigen-specific CD4+ and CD8+ populations can be obtained by stimulating naive T lymphocytes or antigen-specific T lymphocytes with antigen. For example, T cell lines or T cell clones specific for cytomegalovirus antigens can be generated by isolating T cells from a subject infected with cytomegalovirus and stimulating the T cells with the same cytomegalovirus antigen in vitro. Naive T cells may be used. The number of antigens derived from tumor cells used as targets for inducing T cell responses is not particularly limited. In some embodiments, the adoptive cellular immunotherapy compositions of the present invention are useful for treating or alleviating diseases or disorders, including solid tumors, hematological malignancies, breast cancer or malignant melanoma.

[0144] In some embodiments, an isolated T lymphocyte population can be modified with a polynucleotide encoding a CAR as provided herein. In some embodiments, the T cells are obtained from a subject to be treated. In another embodiment, the T lymphocytes are obtained from an allogeneic human donor, preferably a healthy human donor.

[0145] In some embodiments, recombinant infectious viral particles containing a polynucleotide encoding a CAR can be used to deliver genes to T cells. Examples of viral vectors useful in embodiments of the methods and compositions provided herein include viral vectors derived from Simian Virus 40 (SV40), adenovirus, adeno-associated virus (AAV), lentiviral vectors, and / or retroviruses. Examples of techniques for transducing cells, such as T cells, include calcium phosphate transfection, protoplast fusion, electroporation, and infection with viral vectors, such as recombinant adenoviruses, recombinant adeno-associated viruses, and recombinant retroviral vectors. Methods using retroviral and lentiviral vectors allow highly efficient gene transfer into eukaryotic cells. Furthermore, retroviral or lentiviral integration can be controlled, allowing stable integration of one or several copies of new genetic information per cell.

[0146] In some embodiments, a vector comprising a polynucleotide encoding a CAR may contain a positive marker that allows for the selection of cells in vitro. Examples of genes encoding such positive markers include the hygromycin B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, and the adenosine deaminase gene (ADA).

[0147] In some embodiments, CD4+ and CD8+ cells can be modified separately to form a predetermined population using an expression vector encoding a CAR. In some embodiments, the cells thus obtained are further sorted into naive, central memory, and effector cell subpopulations by sorting with cell surface antigens specific to each of these subpopulations, as described above. The CD4+ and CD8+ cell populations may also be selected for their cytokine profile or proliferation activity. For example, CD4+ T lymphocytes can be selected that have enhanced production of cytokines such as IL-2, IL-4, IL-10, TNFα, and / or IFNγ, when stimulated with an antigen, compared to mock-transduced or transduced CD8+ cells. In some embodiments, naive or central memory CD4+ T cells that have enhanced production of IL-2 and / or TNFα are selected. Similarly, CD8+ cells that have enhanced production of IFNγ compared to mock-transduced CD8+ cells are selected.

[0148] In some embodiments, CD4+ and CD8+ cells that proliferate in response to antigen or tumor targets are selected. For example, CD4+ T cells that proliferate more vigorously than mock-transduced or transduced CD8+ cells when stimulated with antigen or tumor targets are selected. In some embodiments, CD4+ and / or CD8+ cells that exhibit cytotoxicity against cells bearing antigen are selected. In embodiments, CD4+ cells are expected to be less cytotoxic than CD8+ cells. In some embodiments, transduced chimeric receptor-expressing T cells that are sustainable in vivo are selected by using established animal models for certain types of cancer. In embodiments, it has been shown that transduced chimeric receptor CD8+ central memory cells with short spacer regions were able to persist in vivo for about 3 days or more, about 10 days or more, about 20 days or more, about 30 days or more, about 40 days or more, or about 50 days or more after introduction into animal models.

[0149] In some embodiments, CD4+ T cells and CD8+ T cells can be further divided into subpopulations, such as, for example, a naive cell population, a central memory cell population, and an effector memory cell population. In some embodiments, the naive CD4+ cells are CD45RO-, CD45RA+, CD62L+, and / or CD4+ T cells. In some embodiments, the central memory CD4+ cells are CD62L- and / or CD45RO-positive. In some embodiments, the effector CD4+ cells are CD62L- and / or CD45RO-positive. Each of these populations may be independently modified with a CAR as described herein.

[0150] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets from CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, central memory T cells (T CM Expression of phenotypic markers of T cells include CD62L, CCR7, CD28, CD3 and / or CD127, and Granzyme B is negative or shows low expression. In some embodiments, central memory T cells are CD45RO+, CD62L+ and / or CD8+ T cells. In some embodiments, effector T cells (T E ) are negative for CD62L, CCR7, CD28 and / or CD127, and positive for granzyme B and / or perforin. In some embodiments, naive CD8+ T lymphocytes are characterized as CD8+, CD62L+, CD45RO+, CCR7+, CD28+, CD127+ and / or CD45RO+. Each of these populations may be independently modified with a CAR.

[0151] In some embodiments, after transduction and / or selection to obtain cells bearing a CAR, the cell population can be expanded in vitro until a sufficient number of cells is obtained for at least one infusion into a human subject, which typically comprises about 10 4 pieces / kg~10 9 In some embodiments, the transduced cells can be cultured in the presence of antigen-bearing cells, anti-CD3 antibodies, anti-CD28 antibodies, IL-2, IL-7, IL-15 and / or IL-21, or combinations thereof. In some embodiments, a subpopulation of CD4+ cells can be combined with a subpopulation of CD8+ cells. In some embodiments, modified naive CD4+ cells or modified central memory CD4+ cells can be combined with modified central memory CD8+ T cells to provide synergistic cytotoxicity against antigen-bearing cells (e.g., tumor cells).

[0152] Methods for Treating or Alleviating a Disease Some embodiments of the methods and compositions provided herein include administering immunotherapy to a subject having a tumor that contains B7H3+ cells to treat or alleviate cancer. Some of these embodiments may include administering to a subject in need of cancer treatment or alleviation an effective amount of cells (such as T cells) that express a CAR that targets the B7H3 antigen. An effective amount of cells may refer to an effective number of cells in a particular cell population.

[0153] As used herein, a "subject" includes humans or non-human mammals, such as dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates, and birds (e.g., chickens), as well as other vertebrates and invertebrates. As used herein, "treat," "treatment," or "treating" may include administering a pharmaceutical composition to a subject for therapeutic purposes, reducing the symptoms or consequences of a disease (e.g., preventing the development of metastases from a primary tumor, reducing the number of tumor cells of a metastatic tumor, or inhibiting the proliferation of tumor cells of a metastatic tumor), and curing a disease (e.g., removing symptoms of a disease, such as eliminating tumor cells of a metastatic tumor in a subject). As used herein, "alleviating" or "alleviating" may include a therapeutic effect that can relieve one or more symptoms of a disease to some extent. As used herein, "prevent", "preventing" and "prevention" may include inhibiting the onset of a disease (e.g., inhibiting metastasis of a primary tumor), preventing major effects that occur before cancer regrowth begins in a subject, and / or inhibiting or reducing the severity of a cancer. As used herein, an "effective amount" may include an amount (e.g., dosage) of a therapeutic compound sufficient to treat a disease.

[0154] In some embodiments, the tumor comprising B7H3+ cells comprises cancers such as malignant melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, non-small cell lung cancer, bladder cancer, renal clear cell carcinoma, and / or glioma. In some embodiments, the tumor comprises a glioma such as oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and / or intrinsic pontine glioma (DIPG). In some embodiments, the tumor comprises a glioblastoma multiforme (GBM). See, e.g., Zhou Z., et al., (2013), J Neurooncol. 111:257-64, which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the CAR-modified T cells provided herein can persist in vivo for at least 3 days or at least 10 days. In some embodiments, the CAR-modified T cells provided herein can expand in vivo for at least 2 or at least 3 generations as measured by dilution of CFSE dye. The expansion and persistence of the CAR-modified T cells can be measured by administering the CAR-modified T cells to an animal model of the disease or disorder and measuring the persistence and / or proliferation ability of the transferred cells. In another embodiment, proliferation and activation can be tested by multiple activation in vitro with antigen-bearing cells.

[0156] In some embodiments, the presence or absence of CAR modified cells in a subject can be determined by obtaining a sample from the subject and detecting the presence or absence of a distinguishing marker, such as the EGFRt distinguishing marker, in the sample.

[0157] In some embodiments, administering the cellular immunotherapy may include administering to the subject a genetically modified helper T lymphocyte cell preparation, the recombinant helper T lymphocyte cell preparation comprising CD4+ T cells having a CAR, such as a CAR that specifically binds to B7H3. In some embodiments, administering the cellular immunotherapy may include administering to the subject a genetically modified cytotoxic T lymphocyte cell preparation, the recombinant cytotoxic T lymphocyte cell preparation comprising CD8+ cells having a CAR, such as a CAR that specifically binds to B7H3. In some embodiments, the CD4+ T helper lymphocytes are selected from naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells prior to the introduction of the CAR. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells, the naive CD4+ T cells comprising CD45RO-, CD45RA+ and / or CD62L+ CD4+ T cells. In some embodiments, the cytotoxic CD8+ T lymphocytes are selected from naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells prior to the introduction of the CAR. In some embodiments, the cytotoxic CD8+ T lymphocytes are central memory T cells, and the central memory T cells include CD45RO+, CD62L+, and / or CD8+ T cells. In some embodiments, the cytotoxic CD8+ T lymphocytes are central memory T cells, and the CD4+ helper T lymphocytes are naive CD4+ T cells.

[0158] The cells produced according to the present invention can be used in methods and compositions for adoptive immunotherapy according to known techniques or modifications thereof as would be readily apparent to one of skill in the art.

[0159] In some embodiments, the cells are formulated by first harvesting the cells from the medium, then washing the cells and concentrating the cells to a therapeutically effective amount in a medium and container system suitable for administration (e.g., a pharma- ceutically acceptable carrier). A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, or lactated Ringer's solution. The infusion medium can be supplemented with human serum albumin, fetal bovine serum, or other human serum components.

[0160] In some embodiments, the therapeutically effective amount of cells in the composition is at least two cell subsets (e.g., one subset of CD8+ central memory T cells and one subset of CD4+ helper T cells). In some embodiments, the therapeutically effective amount of cells in the composition is more typically about 10 2 More than 10 6 10 or less 8 10 or less 9 10 or less 10 The number of cells may be more than 10. The number of cells is determined by the end use of the composition, the type of cells contained in the composition, etc. For example, if cells specific to a particular antigen (e.g., B7H3) are desired, such cells will make up more than 70%, usually more than 80%, more than 85%, and / or 90-95% of the population. In the uses provided herein, the amount of cells is usually 1 L or less (but not 0 L), may be 500 ml or less (but not 0 ml), or even 250 ml or less or 100 ml or less (but not 0 ml). Thus, the density of the desired cells is typically 10 4 More than 10 / ml 7 More than 10 / ml 8 Such clinically relevant numbers of immune cells may be administered in multiple infusions, with the cumulative dose reaching 10 6More than 10 pieces 7 More than 10 pieces 8 More than 10 pieces 9 More than 10 pieces 10 More than 10 pieces 11 More than 10 12 Equivalent to 1 or more.

[0161] In some embodiments, the pharmaceutical composition comprising the cells comprising the CAR provided herein can be administered intravenously, intraperitoneally, intratumorally, intrabone marrow, intralymph nodes, and / or intracerebrospinal fluid. In some embodiments, the cells comprising the CAR provided herein can be delivered to the tumor site. In some embodiments, the pharmaceutical composition comprising the cells comprising the CAR provided herein can be combined with a compound that can direct the cells of the invention to the tumor or immune system compartment but avoid sites such as the lung.

[0162] Combination therapy Some embodiments of the methods and compositions provided herein include treating or alleviating a disease or disorder, such as a cancer having B7H3+ cells, in a subject by immunotherapy in combination with an additional therapeutic regimen. As used herein, combined administration may include administering two or more agents, such as an immunotherapy and an additional therapeutic regimen, to a subject, such that the two or more agents are found in the bloodstream of the subject at the same time, regardless of when and how the two or more agents are actually administered. For example, a first agent, such as a cell expressing a CAR, may be administered to a subject, and an additional therapeutic agent may be administered. In some embodiments, the agents are administered simultaneously. In some such embodiments, combined administration is achieved by combining the agents into a single dosage form. When the agents are combined into a single dosage form, the agents may be physically mixed. In some embodiments, the agents are administered sequentially. In some embodiments, the agents are administered via the same route, such as an intravenous route. In some embodiments, the agents are administered via separate routes, for example, one of the agents is administered orally and the other is administered intravenously.

[0163] In some embodiments, the additional treatment regimen may include radiation therapy. In some embodiments, the additional treatment regimen may include administration of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a cell cycle inhibitor. As used herein, a "cell cycle inhibitor" may include a chemotherapeutic agent that suppresses or inhibits cell division and / or replication. In some embodiments, the cell cycle inhibitor may include chemotherapeutic agents such as doxorubicin, melphalan, roscovitine, mitomycin C, hydroxyurea, 5-fluorouracil, cisplatin, Ara-C, etoposide, gemcitabine, bortezomib, sunitinib, sorafenib, sodium valproate, HDAC inhibitors, dacarbazine, and the like. Further examples of additional chemotherapeutic agents include HDAC inhibitors such as FR01228, trichostatin A, SAHA, and / or PDX101. In some embodiments, the cell cycle inhibitor is a DNA synthesis inhibitor. As used herein, a "DNA synthesis inhibitor" may include a chemotherapeutic agent that suppresses or inhibits DNA synthesis by cancer cells. Examples of DNA synthesis inhibitors include AraC (cytarabine), 6-mercaptopurine, 6-thioguanine, 5-fluorouracil, capecitabine, floxuridine, gemcitabine, decitabine, vidazane, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thiarabine, troxacitabine, sapacitabine or forodesine. Further examples of additional chemotherapeutic agents include FLT3 inhibitors such as semaxanib (SU5416), sunitinib (SU11248), midostaurin (PKC412), lestautinib (CEP-701), tanzutinib (MLN518), CHIR-258, sorafenib (BAY-43-9006) and / or KW-2449. Further examples of additional chemotherapeutic agents include farnesyltransferase inhibitors such as tipifarnib (R115777, Zarnestra), lonafarnib (SCH66336, Sarasar®) and / or BMS-214662.Further examples of additional chemotherapeutic agents include topoisomerase II inhibitors such as epipodophyllotoxins, etoposide, teniposide, anthracyclines, doxorubicin and / or 4-epidoxorubicin. Further examples of additional chemotherapeutic agents include P-glycoprotein modulators such as zosuquidar trihydrochloride (Z.3HCL), vanadate, verapamil, etc. Further examples of additional chemotherapeutic agents include hypomethylating agents such as 5-azacytidine and 2'-deoxyazacytidine.

[0164] kit Some of the embodiments of the methods and compositions provided herein include kits. In some of such embodiments, the kits may include reagents for modifying cells to express a CAR. Some of such embodiments may include an isolated polynucleotide provided herein that encodes a CAR having an antigen binding domain that specifically binds to B7H3. In some embodiments, the kits may include reagents for transducing cells with the isolated polynucleotide that encodes a CAR having an antigen binding domain that specifically binds to B7H3. EXAMPLES

[0165] Example 1 - Activity of B7H3 CAR T cells in vitro and in vivo A viral vector was constructed containing a polynucleotide encoding a CAR targeting human B7H3 using various spacer domains (Figure 1A). Each polynucleotide encoded an antigen-binding domain containing an scFv that specifically binds to human B7H3, one of three spacer domains, a signaling domain containing CD28tm, 41BB and CD3zeta, a T2A skip sequence, and an identification marker containing truncated EGFR (EGFRt). The scFv contained the V H The nucleotide sequence encoding the region and V of SEQ ID NO:16 LThe polynucleotides contained nucleotide sequences encoding the EGFRt region. EGFRt was used for CAR T cell selection and as a tracking marker for CAR T cells in vivo. The spacers used were a short (S) spacer containing a human IgG4 hinge region, a medium (M) spacer containing a human IgG4 hinge region and a CH3 domain, and a long (L) spacer containing a human IgG4 hinge region and a CH2 domain and a CH3 domain. In this example, the long spacer contained a wild-type nucleotide sequence. Thus, each polynucleotide encoded a B7H3 CAR with a short (S) spacer region, a medium (M) spacer region, or a long (L) spacer region (wild-type).

[0166] Using methods substantially similar to those described in Hudecek M, et al., (2013) Clin Cancer Res. 19:3153-64, which is incorporated herein by reference in its entirety, CD8+ T cells were transduced with each polynucleotide encoding the B7H3-CAR to generate B7H3 CAR T cells bearing CARs with a short (S), medium (M) or long (L) spacer region.

[0167] The CAR T cells were subjected to cytotoxicity, cytokine release and proliferation assays using methods substantially similar to those described in Hudecek M et al. (2013). Figure 1B shows the EGFRt discrimination of B7H3 CAR T cells with CARs having short (S), medium (M) or long (L) spacer regions. Figure 1C is a western blot showing CD3ζ expression in B7H3 CAR T cells.

[0168] Cytolysis assays were performed using B7H3 CAR T cells with various B7H3+ cell lines, including human medulloblastoma cell lines (Med411FH and D283), a primitive neuroectodermal tumor cell line (PNET-109FH) and an ependymoma cell line (Epd-110GH). B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions all efficiently lysed B7H3+ cells (Figure 1D). B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions produced cytokines including IFNγ, IL-2 and TNFα in response to stimulation with Med411FH, D283, PNET-109FH or Epd-110GH cells (Figure 1D). B7H3 CAR T cells bearing CARs with a medium-length (M) spacer region showed a trend toward significantly greater cytokine production in response to stimulation with Med411FH cells, PNET-109FH cells, or Epd-110GH cells compared with B7H3 CAR T cells bearing CARs with a short (S) or long (L) spacer region.

[0169] 2×10 B7H3+U87 tumor cells expressing eGFP marker 6The marker was a fusion protein of GFP and ffluc, and the ffluc portion is useful for in vivo imaging. B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions were injected via the tail vein into tumor-free mice or mice transplanted with U87 cells. Bioluminescence imaging was performed according to the method described in Hudecek M et al. (2013). B7H3 CAR T cells bearing CARs with long (L) spacer regions were not used in this experiment because the activity of monocytes remaining in the body of NOD-SCID mice suppressed the activity of CARs containing wild-type long spacers. Figure 1E shows that mice treated with B7H3 CAR T cells bearing CARs with short (S) or medium (M) spacer regions showed a significant reduction in the number of U87 tumor cells and a significant increase in survival rate compared to control mice.

[0170] Example 2 - Activity of B7H3 CAR T cells in vitro and in vivo CD4+ and CD8+ T cells were transduced with polynucleotides encoding B7H3-CAR with a short (S), medium (M) or long (L) spacer region. In this example, a long spacer region containing a double mutant (L235D, N297Q) was used. Expression of various proteins was measured, including EGFRt, immunoglobulins, which are binding partners of protein L, and human Fc, which is a binding partner of anti-Fc (Figure 2A). EGFRt and immunoglobulins were detected in B7H3 CAR T cells containing CARs with a long, medium or short spacer region. The amount of human Fc detected was less in B7H3 CAR T cells containing CARs with a short spacer region than in B7H3 CAR T cells containing CARs with a long spacer region or medium spacer region. This result was consistent with the short spacer not containing a specific Fc moiety. CD3ζ expression was measured in various populations of transduced CD4+ and CD8+ T cells (Fig. 2B).

[0171] Cytolytic assays were performed using B7H3 CAR T cells with various cell lines, including U87 cells (a glioblastoma cell line), modified U87 cells lacking expression of B7H3, Be2 cells (a neuroblastoma cell line), and D283 cells (a medulloblastoma cell line). B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions all efficiently lysed B7H3+ cells (Figure 2C). B7H3 CAR T cells did not show significant cytotoxic effects against modified U87 cells lacking expression of B7H3. B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions all showed activity against U87, Be2, and D283 cells, respectively.

[0172] Cytokine production of B7H3 CAR T cells was measured in vitro in response to stimulation with various cell lines, including Jurkat cells (a T cell line), Jurkat OKT3 cells, U87 cells, modified U87 cells lacking B7H3 expression, Be2 cells, and D283 cells. CD4+ B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions all released greater amounts of cytokines in response to stimulation with U87, Be2, or D283 cells compared to incubation with Jurkat cells, Jurkat OKT3 cells, or modified U87 cells lacking B7H3 expression (Figure 2D). Upon stimulation, CD4+ B7H3 CAR T cells and CD8+ B7H3 CAR T cells bearing CARs with a medium-length (M) spacer region released significantly more cytokines than CD4+ B7H3 CAR T cells and CD8+ B7H3 CAR T cells bearing CARs with short (S) or long (L) spacer regions ( Figure 2D ).

[0173] U87 cells expressing ffluc-eGFP marker were inoculated into NOD-SCID mice. Mice were treated with CD8+ B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions. The number of early detected U87 cells in mice was reduced whether CD8+ B7H3 CAR T cells bearing CARs with short (S), medium (M) or long (L) spacer regions were used. Notably, mice treated with CD8+ B7H3 CAR T cells bearing CARs with short (S) or medium (M) spacer regions showed a more significant and dramatic reduction in the number of early detected U87 cells and an increased survival rate than mice treated with CD8+ B7H3 CAR T cells bearing CARs with long (L) spacer regions (Figure 2E). Thus, B7H3 CAR T cells bearing a CAR with a medium-length (M) spacer region demonstrated significantly greater in vitro and in vivo activity than B7H3 CAR T cells bearing CARs with short (S) or long (L) spacer regions.

[0174] Example 3 - Selection of B7H3 CAR T cells with methotrexate CD4+ T cells were transduced with a viral vector containing a polynucleotide encoding the B7H3 CAR with a medium-length spacer region and a DHFR double mutant (DHFRdm) selection marker. The cells were treated with methotrexate. Expression of EGFRt, human Fab, and human Fc was measured in the various transduced populations over the culture period (Figure 3A). EGFRt, human Fab, and human Fc were expressed in treated cells.

[0175] Cytokine production of B7H3 CAR T cells was measured in vitro upon stimulation with various cell lines, including Jurkat cells (a T cell line), Jurkat OKT3 cells, U87 cells, modified U87 cells lacking B7H3 expression, Be2 cells, and D283 cells. Cytokine production was measured in B7H3 CAR T cells stimulated with U87 cells, Be2 cells, or D283 cells (Figure 3B). Addition of a drug selection gene (DHFRdm) allows enrichment of CAR-expressing T cells in the presence of methotrexate.

[0176] Example 4 - T cells in production run lyse B7H3 positive target cell lines As a target, 51 B7H3 labeled with Cr - (K562 B7H3 KO) cells, control cells (K562 B7H3 KO+OKT3) or various B7H3 + The cell lines (K562 parental, U87MG, U251TMG, SKNBE2 and D283 med cells) were co-cultured with second generation (2GM) B7H3 CAR T cells with a medium-length spacer region for 4 hours. Specific lysis was measured using a scintillation counter (Figure 4). The B7H3 CAR T cells used were a mixture of CD4 T cells (55.7% of total) and CD8 T cells (44.2% of total) with an EGFRt positivity rate of >95%. The histograms inserted in the graph show the positivity rate of B7H3 in each target tumor cell. B7H3 - Little to no specific lysis was detected against the K562 (B7H3 KO) cell line, in contrast to the significant specific lysis observed in the control and experimental cell lines.

[0177] Thus, these data demonstrated that B7H3 CAR T cells specifically lyse B7H3+ control and experimental cell lines.

[0178] Example 5 - T cells in production runs produce cytokines when co-cultured with a B7H3 positive target cell line B7H3 as a target -(K562 B7H3 KO) cells, control cells (K562 B7H3 KO+OKT3) or various B7H3 + Cell lines (K562 parental, U87MG, U251TMG, SKNBE2 and D283 med) were co-cultured with second generation (2GM) B7H3 CAR T cells with medium-length spacer region for 24 hours. The B7H3 CAR T cells used were a mixture of CD4 T cells (55.7% of total) and CD8 T cells (44.2% of total) with EGFRt positivity >95%. The graph (Figure 5) shows the amount of cytokines released by mock or B7H3 CAR T cells when stimulated by co-culture with various target cells of interest. IFNγ, IL-2 and TNFα were measured as cytokines.

[0179] These data suggest that the expression of B7H3 in control cells (K562 B7H3 KO+OKT3) or various B7H3 + T cells co-cultured with cell lines (parental K562 cells, U87MG cells, and SKNBE2 cells) produced each cytokine, but B7H3 - It was shown that T cells co-cultured with (K562 B7H3 KO) cells did not produce any of the cytokines IFNγ, IL-2, or TNFα (FIG. 5).

[0180] Example 6 - T cells in production run can eradicate U87 tumor cells in vivo Mice were inoculated with U87 cells and then treated with CD4+ B7H3 CAR T cells and CD8+ B7H3 CAR T cells produced using the current Tissue Culture and Manufacturing Center (TCPC) T cell manufacturing protocol or the historical PLAT-02 manufacturing protocol. The number of B7H3+ U87 tumor cells detected by ffluc marker in the mice and the survival rate of the mice are shown in Figure 6.

[0181] On day 0, approximately 200,000 GFP:ffluc U87 tumor cells (cJ06289) were injected intracranially into mice (Figure 6). On day 6, flux analysis (by imaging) was performed to confirm the presence of tumor cells in the mouse's intracranial cavity (Figure 6). On day 7, 2 × 10 GFP:ffluc U87 tumor cells with a 1:1 ratio of CD4 T cells to CD8 T cells were injected intracranially into mice (Figure 6). 6 B7H3 CAR T cells were administered at the same intracranial coordinates. Flux analysis (by imaging) and viability were monitored until the end of the study, i.e., day 90 (Figure 6).

[0182] These data demonstrated that both B7H3 CAR T cell populations were able to reduce flux and extend survival in this intracranial U87 model (Figure 6), thus demonstrating that B7H3 CAR T cells can eradicate U87 tumor cells in vivo.

[0183] Example 7 - T cells in production run can eradicate subcutaneous tumor cells in vivo Mice were inoculated with Be2 cells and then treated with CD4+B7H3 and CD8+B7H3 or CD4+CD19 and CD8+CD19 CAR T cells produced using the current Tissue Culture and Manufacturing Center (TCPC) T cell manufacturing protocol or the historical PLAT-02 protocol for CD19 CAR T cell manufacturing. The number of B7H3+ Be2 tumor cells detected by ffluc marker in the mice and the survival rate of the mice are shown in Figure 7.

[0184] In this model, approximately 4.3 × 10 6 CD19t GFP:ffluc Be2 tumor cells (cJ06713) were injected subcutaneously on day 0 (Figure 7). On day 4, flux analysis (by imaging) was performed to confirm the presence of tumor cells in the mouse (Figure 7). On day 5, approximately 20 × 10 6CAR T cells were administered intravenously (Figure 7). Flux analysis (by imaging) and viability were monitored until the end of the study, i.e., day 90 (Figure 7).

[0185] These data demonstrated that both B7H3 and CD19 CAR T cells were able to reduce flux and extend survival in this subcutaneous tumor Be2 model (Figure 7). Thus, these data demonstrated that both B7H3 and CD19 CAR T cells can eradicate subcutaneous tumor cells in vivo.

[0186] Example 8 - T cells in production run can eradicate subcutaneous tumor cells in vivo Mice were inoculated with Be2 cells and then treated with CD4+B7H3 T cells and CD8+ B7H3 CAR T cells produced using the current Tissue Culture and Manufacturing Center (TCPC) T cell manufacturing protocol or the historical PLAT-02 protocol for T cell manufacturing. The number of B7H3+ Be2 tumor cells detected by ffluc marker in the mice and the survival rate of the mice are shown in Figure 8.

[0187] In this model, approximately 5 × 10 6 CD19t GFP:ffluc Be2 tumor cells (cJ06713) were injected subcutaneously on day 0 (Figure 8). On day 6, flux analysis (by imaging) was performed to confirm the presence of tumor cells in the mouse (Figure 8). On day 5, 30 × 10 6 B7H3 CAR T cells were administered intravenously (Figure 8). Flux analysis (by imaging) and viability were monitored until the end of the study, i.e., up to day 75 (Figure 8).

[0188] These data demonstrated that both B7H3 CAR T cell lots were able to reduce flux and extend survival in this subcutaneous tumor Be2 model (Figure 8). Thus, these data demonstrated that both B7H3 CAR T cell lots were able to eradicate subcutaneous tumor cells in vivo.

[0189] Example 9 - CD8+ B7H3 CAR T cells can eradicate U251T glioma cells in vivo Figure 9 shows the number of B7H3+ U251T tumor cells detected by ffluc marker in mice inoculated with U251T cells and treated with CD8+ B7H3 CAR T cells, and the survival rate of these mice.

[0190] In this model, approximately 200,000 GFP:ffluc U251T tumor cells (cJ06194) were injected intracranially on day 0 (Figure 9). On day 6, flux analysis (by imaging) was performed to confirm the presence of tumor cells in the mouse skull (Figure 9). On day 7, approximately 2 × 10 6 Mock or B7H3 CAR T cells were administered at the same intracranial coordinates. Flux analysis (by imaging) and viability were monitored until the end of the study, i.e., day 90 (Figure 9).

[0191] These data demonstrated that the use of B7H3 CAR T cell populations was able to reduce flux and extend survival in this intracranial U251T model (Figure 9). Thus, these data demonstrated that CD8+ B7H3 CAR T cells can eradicate U251T glioma cells in vivo.

[0192] Example 10 - CD8+ B7H3 CAR T cells can eradicate primary tumor cells of diffuse intrinsic pontine glioma (DIPG) in vivo Figure 10 shows the number of B7H3+ DIPG tumor cells detected by ffluc marker in mice inoculated with DIPG cells and treated with CD8+ B7H3 CAR T cells, and the survival rate of these mice.

[0193] In this model, approximately 0.2 × 106 mCherry:ffluc Pbt09mcl DIPG tumor cells were injected intracranially on day 0 (Figure 10). On day 39, flux analysis (by imaging) was performed to confirm the presence of tumor cells in the mouse's intracranial cavity (Figure 10). On day 40, approximately 2x10 6 CD8 bulk Mock cells or CD8 bulk B7H3 CAR T cells were administered at the same coordinates (Figure 10). Flux analysis (by imaging) and viability were monitored until the end of the study, i.e., day 90 (Figure 10). Although the study was terminated before the Mock group died of tumor, treatment with B7H3 CAR T cells was able to reduce the flux to baseline compared to the Mock-treated group. DIPG is a rare type of tumor and is currently not a target of cellular immunotherapy approaches.

[0194] These data demonstrated that this B7H3 CAR T cell population was able to reduce flux and extend survival in this intracranial DIPG model (Figure 10). Thus, these data demonstrated that CD8+ B7H3 CAR T cells can eradicate primary DIPG tumor cells in vivo.

[0195] As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by" and is open-ended and inclusive and does not exclude additional elements or steps not described herein.

[0196] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and equipment. Such modifications will be readily apparent to those skilled in the art from practice of the invention disclosed herein or from consideration of this disclosure. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but will encompass all modifications and other aspects which fall within the true scope and spirit of the invention.

[0197] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and journal articles, are hereby incorporated by reference in their entirety and made a part of this specification. In the event that any reference, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter.

Claims

[Claim 1] The invention described herein.

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