CN117120077A - Cell therapy compositions and methods for modulating TGF-B signaling - Google Patents

Cell therapy compositions and methods for modulating TGF-B signaling Download PDF

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CN117120077A
CN117120077A CN202280014920.1A CN202280014920A CN117120077A CN 117120077 A CN117120077 A CN 117120077A CN 202280014920 A CN202280014920 A CN 202280014920A CN 117120077 A CN117120077 A CN 117120077A
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cells
car
tgf
cell
cancer
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C·库恩
G·夏皮罗
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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Priority claimed from PCT/IB2022/000063 external-priority patent/WO2022172085A2/en
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Abstract

Methods of using polypeptides to modulate transforming growth factor-beta (tgfβ) signaling (e.g., tgfβ receptors, antibodies or antigen binding fragments thereof that specifically bind to tgfβ or tgfβ receptors) are provided. Compositions comprising the antibodies or fragments thereof and methods of using the compositions in the treatment of diseases involving tgfβ activity are provided. Nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions comprising these antigen-binding agents and fragments thereof are also disclosed. The application also provides methods of treatment for using modulators of tgfβ signaling.

Description

Cell therapy compositions and methods for modulating TGF-B signaling
Cross Reference to Related Applications
The present application claims priority from U.S. provisional application No. 63/149,628 filed on day 2021, 2 and 15 and U.S. provisional application No. 63/306,836 filed on day 2022, 2 and 04, the disclosures of each of which are hereby incorporated by reference in their entirety.
Background
Immunotherapy using engineered cells targeting cancer specific antigens has shown efficacy in the treatment of some cancers. However, malignant cells will adapt to create an immunosuppressive microenvironment to protect them from immune recognition and elimination. High tgfβ levels in the tumor microenvironment may promote the maintenance and progression of some types of cancer cells. The tumor microenvironment presents significant challenges for therapeutic approaches involving stimulation of immune responses, such as in the case of targeted cell therapies. Thus, there is a need for novel therapeutic strategies for treating cancer.
Disclosure of Invention
The invention provides, inter alia, a novel system for modulating tgfβ signaling for use in the treatment of cancer (e.g., solid tumors). The present invention is based in part on the following findings: modulation of transforming growth factor beta (TGF-beta) signaling may enhance adoptive cell therapy methods, such as targeted engineered Chimeric Antigen Receptor (CAR) therapies. Modulation of TGF-beta signaling, e.g., effected by an antibody system (e.g., anti-tgfβ or anti-tgfβr), antigen binding fragments of TGF- βr2, or recombinant extracellular domains as described herein, would reduce the immunosuppressive microenvironment in the tumor and enhance the efficacy of immunotherapy.
T cell-based immunotherapy has become a new front of synthetic biology; multiple promoters and gene products are designed to divert these high-efficiency cells into tumor microenvironment, where T cells can bypass negative regulatory signals and mediate efficient tumor killing. Unwanted T cells were eliminated by drug-induced dimerization of inducible caspase 9 (caspase 9) with AP1903, indicating a method by which the powerful switch controlling the T cell population could be pharmacologically activated (Di Stasi A et al, N Engl J Med.2011;365 (18): 1673-83). Thus, while CARs appear to trigger T cell activation in a manner similar to endogenous T cell receptors, the major impediment to clinical use of this technology to date has been limited in the in vivo expansion of car+ T cells, rapid disappearance of cells after infusion, and disappointing clinical activity. Thus, there is an urgent need in the art to find novel compositions and methods for treating cancer in a manner that can exhibit specific and potent anti-tumor effects without undesired effects (i.e., high toxicity, insufficient efficacy).
The present invention addresses these needs by providing an immune modulation system comprising a CAR and a tgfβ signaling pathway modulator expressed in immune cells (e.g., T cells). Compositions and methods of treatment comprising the immune modulating system are useful for treating cancer and other diseases and/or conditions. In particular, the invention provides engineered immune cells expressing an armored CAR that are useful for treating diseases, disorders, or conditions associated with deregulation of expression of tgfβ (e.g., cancer, solid tumors). Armored CAR T cells co-expressing tgfβ modulators exhibit high surface expression of CARs on transduced T cells, and enhanced cytolysis of cancer cells. Accordingly, the present invention provides methods and compositions for enhancing immune responses to cancers and pathogens using immune modulating systems (e.g., engineered CAR T cells) comprising polypeptides that modulate TGF-b signaling.
The present invention provides, in part, improved CAR polypeptides comprising tgfβ signaling pathway modulators, nucleic acid molecules encoding such polypeptides, cells (e.g., T cells) genetically modified to express the improved CARs, and methods of using the modified cells in adoptive cell therapies for treating cancer (e.g., solid tumor cancer).
In some embodiments, the invention provides CAR-T cells modified to express a tgfp signaling pathway modulator (also referred to herein as "tgfp-armored CAR-T cells") such that when administered to a subject in need thereof, the cells are capable of eliciting an immune response in the subject relative to CAR-T cells that do not express a tgfp signaling pathway modulator (also referred to herein as "unarmored CAR-T cells").
In some aspects, the invention provides an immunoreactive cell (e.g., a T cell) bearing an antigen receptor, which may be a Chimeric Antigen Receptor (CAR), and which comprises a polypeptide that modulates TGF-b signaling. These engineered immunoreactive cells (e.g., CAR-T cells) are antigen-directed and may be resistant to immunosuppression and/or have enhanced immune activation properties.
In one aspect, the invention provides a population of genetically engineered T cells comprising a Chimeric Antigen Receptor (CAR) that recognizes a cancer-associated antigen and a tgfβ signaling pathway modulator.
In some embodiments, the cell population comprises a CAR that recognizes an antigen selected from the group consisting of: ADGRE2, CLEC12, CAIX, CEA, CD, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, CEACAM 5, tight junction protein (Claudin) 18.2, EGP-2, EGP-40, epCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY 2C), GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, leY, LI cell adhesion molecule, MAGE-AI, MUC1, MUC13, mesothelin, NKG2D ligand, NY-ES0-1, tumor embryo antigen (h 5T 4), PSCA, PSMA, PTK, R1, TAG-72, TROP2, VEGF-R2 and WT 1.
In some embodiments, the population of cells comprises a CD19 CAR or a GCC CAR.
In some embodiments, the cell population comprises a tgfβ signaling pathway modulator that binds to tgfβ or a tgfβ receptor.
In some embodiments, the cell population comprises a tgfβ signaling pathway modulator comprising an amino acid sequence selected from table 1.
In some embodiments, the population of cells is autologous.
In some embodiments, the population of cells is allogeneic.
In some embodiments, the cell population is primary cells. In some embodiments, the cell population is derived from induced pluripotent stem cells (ipscs).
In some embodiments, the cell population is genetically modified using a vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator.
In some embodiments, the cell population is genetically modified using two vectors, a first vector comprising a nucleic acid encoding a CAR polypeptide and a second vector comprising a nucleic acid encoding a tgfβ signaling pathway modulator.
In some embodiments, the cell population is genetically modified using Crispr. In some embodiments, the cell population is genetically modified using retroviral transduction (including g-retroviruses), lentiviral transduction, transposon and transposase (sleep Beauty and PiggyBac systems), messenger RNA transfer mediated gene expression, gene editing (gene insertion or gene deletion/disruption), CRISPR-Cas9, ZFN (zinc finger nuclease), or TALEN (transcription activator-like effector nuclease) systems.
In some embodiments, the cell population comprises a CAR comprising an intracellular signaling domain selected from the group consisting of: CD3 zeta-chain, CD97, 2B4, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, DAP10, DAP12, CD28 signaling domain, or combinations and variations thereof.
In some embodiments, the population of cells comprises a CAR comprising a transmembrane domain derived from a transmembrane domain selected from the group consisting of: CD3, CD8, CD28, OX40, CD27, 4-1BB, DAP10, DAP12, or combinations thereof.
In one aspect, the invention provides a vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator.
In some embodiments, the vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator comprises an internal ribosome entry site.
In some embodiments, the vector further comprises a 2A ribosomal sequence.
In one aspect, the invention provides an immune cell modified with a vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator.
In some embodiments, the immune cell is a T cell.
In one aspect, the invention provides a method of modulating an immune response in a host, the method comprising administering to the host a population of genetically engineered T cells comprising a Chimeric Antigen Receptor (CAR) that recognizes a cancer-associated antigen and a tgfβ signaling pathway modulator, wherein modulation of the immune response comprises one or more of the following by a host immune cell: increase ifnγ production; increase IL-2 production; increasing antigen presentation; and increase proliferation.
In one aspect, the invention provides a pharmaceutical composition comprising a population of genetically engineered T cells comprising a Chimeric Antigen Receptor (CAR) that recognizes a cancer-associated antigen and a tgfβ signaling pathway modulator.
In one aspect, the invention provides a method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of genetically engineered T cells comprising a Chimeric Antigen Receptor (CAR) that recognizes a cancer-associated antigen and a tgfβ signaling pathway modulator.
In some embodiments, the cancer is selected from the group consisting of: leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia), heavy chain disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, solitary tumor, angiosarcoma, endotheliosarcoma, lymphosarcoma, lymphangioendothelioma, mesothelioma, ewing's tumor (Ewing's tumor), leiomyosarcoma rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, cyst gland carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms ' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, colorectal cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngeal tumor, ependymoma, pineal tumor, angioblastoma, auditory neuroma, oligodendroglioma, neurosheath tumor, meningioma, melanoma, neuroblastoma, retinoblastoma, and metastases thereof.
In one aspect, the invention provides an immune modulatory system comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR); and nucleic acid sequences encoding polypeptides that modulate TGF-b signaling (e.g., tgfβ signaling modulators).
In some embodiments, the polypeptide that modulates TGF-b signaling comprises a variable heavy chain (vH).
In some embodiments, the polypeptide that modulates TGF-b signaling comprises a variable heavy chain (vH) and a variable light chain (vL).
In some embodiments, the polypeptide that modulates TGF-b signaling comprises an antigen binding molecule selected from the group consisting of: igA antibodies, igG antibodies, igE antibodies, igM antibodies, bispecific or multispecific antibodies, fab fragments, fab ' fragments, F (ab ') 2 fragments, fd ' fragments, fd fragments, isolated CDRs, or collections thereof; single chain variable fragments (scFv), polypeptide-Fc fusions, single domain antibodies (sdabs), camelized antibodies; masking antibodies, small modular immunopharmaceuticals ("SMIPsTM"), single chain, tandem diabodies, VHH, anti-cargo proteins (anti-antibodies), nanobodies, humanized antibodies (humabody), minibodies, biTE, ankyrin (ankyrin) repeat proteins, DARPIN, avimer, DART, TCR-like antibodies, adnectin, affilin, penetrating antibodies (Trans-body); affinity antibody (Affibody), trimerX, miniprotein, fynomer, centyrin; and KALBITOR, or a fragment thereof.
In some embodiments, the polypeptide that modulates TGF-b signaling comprises a single chain variable fragment (scFv). In some embodiments, the polypeptide that modulates TGF-b signaling comprises a single domain antibody (sdAb). In some embodiments, the polypeptide that modulates TGF-b signaling comprises a heavy chain-only antibody.
In some embodiments, the polypeptide that modulates TGF-b signaling comprises an amino acid sequence selected from table 1.
In some embodiments, the polypeptide that modulates TGF-b signaling comprises a dimeric antigen binder.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to TGF-b.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to a TGF-b receptor.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to TGF-b receptor 2 (TGF-bR 2).
In some embodiments, the polypeptide that modulates TGF-b signaling comprises TGF-b receptor 2 (TGF-bR 2) or a fragment thereof.
In some embodiments, the polypeptide that modulates TGF-b signaling comprises an extracellular domain of TGF-bR2 (TGF-bR 2).
In some embodiments, the CAR binds to an antigen selected from the group consisting of: ADGRE2, CLEC12, CAIX, CEA, CD, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, CEACAM 5, claudin 18.2, EGP-2, EGP-40, epCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY 2C), GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, leY, LI cell adhesion molecule, MAGE-AI, MUC1, MUC13, mesothelin, NKG2D ligand, NY-ES0-1, tumor embryo antigen (h 5T 4), PSCA, PSMA, PTK, ROR1, TAG-72, TROP2, VEGF-R2 and WT-1.
In some embodiments, the CAR binds to CD19 or GCC.
In some embodiments, the CAR comprises an intracellular signaling domain selected from the group consisting of: CD3 zeta-chain, CD97, 2B4, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, DAP10, DAP12, CD28 signaling domain, or combinations and variations thereof.
The immune modulatory system of any one of the preceding claims, wherein said CAR comprises a transmembrane domain derived from a transmembrane domain selected from the group consisting of: CD3, CD8, CD28, OX40, CD27, 4-1BB, DAP10, DAP12, or combinations thereof.
In certain embodiments, the modified CD3z polypeptide lacks all or part of an immunoreceptor tyrosine-based activation motif (ITAM), wherein the ITAM is ITAM1, ITAM2, and ITAM3. In certain embodiments, the modified CD3z polypeptide further lacks all or part of the basic-rich extension (BRS) region, wherein the BRS regions are BRS1, BRS2, and BRS3.
In one aspect, the invention provides a nucleic acid comprising an immune modulatory system as described herein, wherein the sequence encoding the Chimeric Antigen Receptor (CAR) and the sequence encoding the polypeptide which modulates TGF-b signaling are present on a single construct.
In some embodiments, the sequence encoding the Chimeric Antigen Receptor (CAR) and the sequence encoding the polypeptide that modulates TGF-b signaling are present on different constructs.
In one aspect, the invention provides a vector comprising a nucleic acid encoding an immune modulating system as described herein.
In some embodiments, the vector comprises an Internal Ribosome Entry Site (IRES).
In some embodiments, the vector comprises a 2A ribosomal sequence. In some embodiments, the 2A ribosomal sequence is P2A or T2A.
In one aspect, the invention provides an immunoreactive cell comprising an immunomodulatory system described herein.
In one aspect, the invention provides an immunoreactive cell comprising: targeting agents specific for tumor-associated antigens or stress ligands, and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In some embodiments, the targeting agent specifically binds to a stress ligand selected from the group consisting of: MIC-A, MIC-B, ULBP1-6;
in one aspect, the invention provides an immunoreactive cell comprising: chimeric Antigen Receptor (CAR); and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In some embodiments, the CAR and the nucleic acid encoding a polypeptide that modulates TGF-b signaling are provided on the same polynucleotide.
In some embodiments, the CAR and the nucleic acid encoding a polypeptide that modulates TGF-b signaling are provided on separate polynucleotides.
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling is secreted by the cell.
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises a variable heavy chain (vH).
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises a variable heavy chain (vH) and a variable light chain (vL).
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises a single chain variable fragment (scFv).
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises a dimeric antigen binder.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to TGF-b.
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises TGF-b receptor 2 (TGF-bR 2) or a fragment thereof.
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises an extracellular domain of TGF-bR 2.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to a TGF-b receptor.
In some embodiments, the polypeptide that modulates TGF-b signaling binds to TGF-b receptor 2 (TGF-bR 2).
In some embodiments, the immunoreactive cell comprises a CAR expressed from a vector, an engineered mRNA, or integrated into the chromosome of a host cell. In some embodiments, the CAR-encoding sequence is integrated into the host cell chromosome using an endonuclease. In some embodiments, the sequence encoding the CAR is integrated into the host cell chromosome using Crispr/Cas9, cas12a, or Cas 13.
In some embodiments, the immunoreactive cells comprise a recombinant polypeptide that modulates TGF-b signaling, expressed from a vector, engineered mRNA, or integrated into the host cell chromosome. In some embodiments, the sequence encoding a polypeptide that modulates TGF-b signaling is integrated into the host cell chromosome using Crispr/Cas9, cas12a, or Cas 13.
In some embodiments, the immunoreactive cells are selected from the group consisting of: t cells, natural Killer (NK) T cells, γδ T cells, cytotoxic T Lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, B cells, macrophages, and pluripotent stem cells from which lymphoid cells may be differentiated (e.g., NK or T cells derived from ipscs).
In some embodiments, the immunoreactive cells are engineered autologous cells. In some embodiments, the immunoreactive cells are engineered allogeneic cells.
In some embodiments, the immunoreactive cell comprises a CAR that binds to a tumor antigen selected from the group consisting of: ADGRE2, CLEC12, CAIX, CEA, CD, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, CEACAM 5, claudin 18.2, EGP-2, EGP-40, epCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY 2C), GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, leY, LI cell adhesion molecule, MAGE-AI, MUC1, MUC13, mesothelin, NKG2D ligand, NY-ES0-1, tumor embryo antigen (h 5T 4), PSCA, PSMA, PTK, ROR1, TAG-72, TROP2, VEGF-R2 and WT-1.
In some embodiments, the CAR binds to CD19 or GCC. In some embodiments, the CAR binds to GCC.
In some embodiments, the CAR comprises an intracellular signaling domain derived from: CD3 ζ, CD97, 2B4, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, DAP10, DAP12, CD28 signaling domains, or combinations and variations thereof.
In some embodiments, the CAR comprises a transmembrane domain derived from a transmembrane domain selected from the group consisting of: CD3, CD8, CD28, OX40, CD27, 4-1BB, DAP10, DAP12, or combinations thereof.
In certain embodiments, the modified CD3z polypeptide lacks all or part of an immunoreceptor tyrosine-based activation motif (ITAM), wherein the ITAM is ITAM1, ITAM2, and ITAM3. In certain embodiments, the modified CD3z polypeptide further lacks all or part of the basic-rich extension (BRS) region, wherein the BRS regions are BRS1, BRS2, and BRS3.
In some embodiments, the immunoreactive cells comprise a chimeric co-stimulatory receptor (CCR). In some embodiments, the CAR comprises a co-stimulatory domain. In some embodiments, the CAR does not comprise an intracellular signaling domain. In some embodiments, the CAR does not comprise a CD3z domain.
In some embodiments, the recombinant polypeptide that modulates TGF-b signaling enhances the immune response of an immunoreactive cell.
In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of the immune modulating system described herein.
In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of a nucleic acid sequence encoding an immune modulatory system as described herein.
In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of a vector encoding an immune modulation system as described herein.
In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of an immunoreactive cell described herein.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
In one aspect, the invention provides a kit for treating cancer, the kit comprising immunoreactive cells comprising a Chimeric Antigen Receptor (CAR); and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In some embodiments, the kit comprises a nucleic acid or vector encoding an immune modulation system described herein.
In one aspect, the invention provides a method of treating or preventing cancer or metastasis thereof in a subject, the method comprising administering an effective amount of an immunoreactive cell comprising a Chimeric Antigen Receptor (CAR); and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In some embodiments, the compositions described herein are useful for treating hematopoietic cancers. In other embodiments, the compositions described herein can be used to treat solid tumor cancers.
In some embodiments, the cancer is selected from the group consisting of: leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, fahrenheit macroglobulinemia, heavy chain disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, solitary tumor, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, colorectal cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngeal tube tumor, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, neurosphingoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
In some embodiments, the method further comprises administering a second therapeutic agent to the subject.
In some embodiments, the second therapeutic agent is administered to the subject in an systemic manner.
In some embodiments, the second therapeutic agent is administered separately from the CAR and the nucleic acid encoding the recombinant polypeptide that modulates TGF-b signaling
In some embodiments, the second therapeutic agent targets PD1/PD-L1, CXCR2, and/or IL-15.
In some embodiments, the second therapeutic agent is a PD1/PD-L1 inhibitor.
In one aspect, the invention provides a method of modulating immune cell activity, the method comprising administering a nucleic acid encoding a Chimeric Antigen Receptor (CAR); and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In one aspect, the invention provides a method of modulating the activity of a Chimeric Antigen Receptor (CAR), the method comprising administering a nucleic acid encoding a Chimeric Antigen Receptor (CAR); and nucleic acids encoding recombinant polypeptides that modulate TGF-b signaling.
In one aspect, the invention provides a method of reducing tumor burden in a subject, the method comprising administering an effective amount of an immune modulating system comprising a nucleic acid, vector, or immunoreactive cell as described herein.
In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces tumor size. In some embodiments, the method excludes a tumor in the subject.
In one aspect, the invention provides a method of increasing immune-activated cytokine production in response to cancer cells in a subject, the method comprising administering to the subject an immune modulating system comprising a nucleic acid, vector, or immunoreactive cell described herein.
In one aspect, the invention provides a method for producing an antigen-specific immunoreactive cell, the method comprising introducing into the immunoreactive cell a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and a nucleic acid encoding a recombinant polypeptide that modulates TGF-b signaling.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
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The drawings included herein, which are comprised of the following figures, are for illustrative purposes only and are not limiting.
FIGS. 1A-1E show exemplary expression of a CAR and a TGF-beta signaling modulator in an immunoreactive cell (e.g., a transduced T cell). Fig. 1A illustrates a lymphocyte population, fig. 1B illustrates a single cell population, fig. 1C depicts a live cd3+ cell population, and fig. 1D shows exemplary flow cytometry results evaluating CAR expression in TGF- β expressing armored human CAR-T cells. FIG. 1E depicts histograms showing transduction efficiency expressed in% of live cells positive for CAR staining using non-armored cells transduced with CD19CAR alone, and CD19 CAR-T cells armored with TGFb scFv VH-VL1 (SEQ ID NO: 1), TGFb scFv VH-VL2 (SEQ ID NO: 2), TGFb scFv VL-VH (SEQ ID NO: 3), TGFbR2 scFv VH-VL (SEQ ID NO: 4), TGFbR2 scFv VL-VH (SEQ ID NO: 5), mGFbR 2 VH1 (SEQ ID NO: 6) and hTGFbR2 VH1 (SEQ ID NO: 8), or non-transduced cells.
The graphs of fig. 2A-2B demonstrate exemplary in vivo killing assay results for cd19+ Raji cells (fig. 2A) and CD19ko Raji cells (fig. 2B) for immunoreactive cells that co-express an anti-CD 19 CAR and a modulator of TGF- β signaling using effectors that are targets relative to: non-armored cells transduced with CD19 CAR alone, and CD19 CAR-T cells armored with TGFb scFv VH-VL1 (SEQ ID NO: 1), TGFb scFv VH-VL2 (SEQ ID NO: 2), TGFb scFv VL-VH (SEQ ID NO: 3), TGFbR2 scFv VH-VL (SEQ ID NO: 4), TGFbR2 scFv VL-VH (SEQ ID NO: 5), mTGFbR2 VH1 (SEQ ID NO: 6) and hTGFbR2 VH1 (SEQ ID NO: 8), or non-transduced cells.
Fig. 3A depicts a histogram showing exemplary ELISA results demonstrating TGF- β binder secretion by human CAR-T cells, and fig. 3B depicts a histogram showing exemplary ELISA results demonstrating TGF- β R2 binder secretion by human CAR-T cells, and their ability to bind to cognate antigen.
FIG. 4 depicts histograms showing exemplary luciferase assay results evaluating inhibition of TGF-beta signaling by supernatants from CAR-T cells of secretion constructs TGFb scFv VH-VL1 (SEQ ID NO: 1), TGFb scFv VH-VL2 (SEQ ID NO: 2), TGFb scFv VL-VH (SEQ ID NO: 3), TGFbR2 scFv VH-VL (SEQ ID NO: 4), TGFbR2 scFv VL-VH (SEQ ID NO: 5), mTGFbR2 VH1 (SEQ ID NO: 6) and hTGFbR2 VH1 (SEQ ID NO: 8).
FIG. 5A shows a histogram depicting the results of an exemplary luciferase assay evaluating the inhibition of TGF-beta signaling by supernatants from CAR-T cells secreting TGFb-scFv VH-VL 1G 4S dimer (SEQ ID NO: 17), TGFb-scFv VH-VL1 2xG4S dimer (SEQ ID NO: 18), TGFb-scFv VH-VL1 minibody (SEQ ID NO: 21), TGFb-scFv VH-VL1 minibody+hinge (SEQ ID NO: 19). FIG. 5B shows a schematic of an exemplary TGF-beta modulator designed and using a luciferase reporter assay to screen for secretion of multimeric binders to TGF-beta. FIG. 5C shows a schematic representation of an exemplary TGF-beta modulator comprising a VHH binding domain.
FIG. 6A shows a histogram depicting exemplary luciferase assay results evaluating the relative blocking activity of armored CAR T cells co-expressing monomeric TGFb scFv VH-VL1 (SEQ ID NO: 1) and dimeric TGFb-scFv VH-VL 1G 4S dimer (SEQ ID NO: 17) binders compared to non-armored cells expressing only CAR. FIG. 6B shows a histogram depicting the results of an exemplary luciferase assay that evaluates the relative blocking activity of TGF-beta R2VHH and scFv monomer and dimer constructs. Non-armored CAR-T cells, mTGFbR 2VH 2 monomer, mTGFbR 2VH 2G 4S dimer, mTGFbR 2VH 2G 4S trimer, httgfbr 2VH 2 monomer, httgfbr 2VH 2G 4S dimer, httgfbr 2VH 3 monomer, httgfbr 2VH 3G 4S dimer, httgfbr 2scFv VH-VL monomer, httgfbr 2scFv VH-VL G4S dimer.
The histograms depicted in fig. 7A and 7B depict exemplary ELISA results, demonstrating that exemplary TGFb modulators bind to human TGFbR2 (fig. 7A) but not to mouse TGFbR2 (fig. 7B). Non-armored CAR-T cells, mTGFbR2VH2 monomer, mTGFbR2VH 2G 4S dimer, mTGFbR2VH 2G 4S trimer, httgfbr 2VH2 monomer, httgfbr 2VH 2G 4S dimer, httgfbr 2VH 3 monomer, httgfbr 2VH 3G 4S dimer, httgfbr 2 scFv VH-VL monomer, httgfbr 2 scFv VH-VL G4S dimer.
Fig. 8A shows an exemplary injection schedule to evaluate tumor growth of EMT6-hCD19-Fluc tumor cells as described in example 6. Fig. 8B shows exemplary tumor volume changes over time in mice receiving CAR-T cells that secrete TGF- β binders relative to non-armored CAR-T or non-transduced CAR-T cells. Figure 8C demonstrates exemplary liver metastasis in mice treated with CAR-T cells that secrete TGF- β binders relative to non-armored or non-transduced CAR-T cells. Figure 8D presents exemplary lung metastasis in mice treated with CAR-T cells that secrete TGF- β binders relative to non-armored or non-transduced CAR-T cells. Fig. 8E presents exemplary imaging results of luciferase-expressing tumor cells in liver and lung tissue.
Fig. 9A and 9B depict histograms showing exemplary SBE-Luc TGF-B report assay results comparing armored mouse CAR-T cells from secreting different TGF-B ligand traps (TGF-B scFv VH-VL1 to TGFbR 2ECD monomers, homodimers (fig. 9A) and heterodimers (fig. 9B)) with supernatants from unarmored CAR-T cells.
FIG. 10A shows an exemplary injection schedule to evaluate tumor growth of EMT6-hCD19-Fluc tumor cells. Fig. 10B shows exemplary tumor volume changes over time in mice receiving either non-transduced T cells or non-armored CAR-T cells (CAR-T cells that do not co-express a tgfβ signaling modulator). Fig. 10C shows exemplary tumor volume changes over time in mice receiving either armored CAR-T cells or non-armored CAR-T cells that co-express tgfbr1+2ECD dimers (CAR-T cells that do not co-express tgfβ signaling modulators). Fig. 10D shows exemplary tumor volume changes over time in mice receiving either systemic anti-TGFb antibody (1D 11) or non-armored CAR-T cells (CAR-T cells that do not co-express tgfβ signaling modulators).
Fig. 11 depicts a graph showing exemplary tumor volume changes over time in mice developed from MC38 cells expressing CD 19. Mice received either non-transduced T cells, non-armored anti-CD 19 CAR-T cells, or CAR-T cells that secrete an inhibitory binding partner for anti-TGF-b (TGF-b scFv VH-VL 1).
FIG. 12 depicts a graph showing an exemplary RNA Seq analysis that demonstrates enhanced activation of host immune responses by CAR-T cells secreting binders to anti-TGF-b (TGF-b scFv VH-VL1 (SEQ ID NO: 1)).
FIG. 13 shows exemplary biomarker scores for tumor infiltrating T cells (CD3d+, CD3e+, CD3g+), CD8+ T cells (CD8a+) and cytotoxic T cells (GzmB+) in tumors of mice receiving CAR-T cells that secrete TGF-b scFv VH-VL1 (SEQ ID NO: 1).
FIG. 14 shows an exemplary single sample Gene Set Enrichment Analysis (GSEA) with enrichment scores exhibiting an increase in T cell and IFNg characteristics in mice tumors receiving CAR-T cells secreting TGF-b scFv VH-VL1 (SEQ ID NO: 1).
FIG. 15 shows exemplary surface marker assays including TCRa/b, CD8a, CD4, CD25, CD62L, CD11b, gr1, CD11c, CD45.1 and CD45 in mice receiving non-transduced control T cells, non-armored CD19 CAR-T cells, or CAR-T cells secreting anti-TGF-b scFv VH-VL1 (SEQ ID NO: 1) monomers.
Fig. 16A and 16B are graphs depicting exemplary in vivo analyses of GSU allograft models demonstrating improved function of human GCC-CAR-T cells armored with anti-TGF-B or anti-TGFbR 2 blocking antibodies.
Figures 17A-17D show tumor and/or plasma concentrations of TGFb modulator secreted by anti-GCC CAR-T cells co-expressing TGF-b scFv VH-VL1 and TGFbR2VHH, as determined using an anti-Flag immunocapture LC/MS assay.
Fig. 18A-18C are graphs depicting exemplary in vitro killing assay results in HT29-GCC positive cells using non-armored anti-GCC CAR-T cells, anti-TGFbR 2VHH monomelic armored anti-GCC CAR-T cells, and anti-TGFbR 2VHH dimer armored anti-GCC CAR-T cells in the absence of TGFb (fig. 18A) and in the presence of TGFb (fig. 18B). Figure 18C shows CAR T cell proliferation in the presence and absence of TGFb.
FIG. 19 depicts exemplary flow cytometry results demonstrating PD-1/Lag3 expression on cells following repeated antigen stimulation.
FIGS. 20A-20C depict xenograft models of GCC expressing cells GSU (FIG. 20A), HT55 (FIG. 20B), and MDA-MB-231-FP4 Luc (FIG. 20C) treated with both armored and non-armored CAR-T cells and CAR-T cells expressing dominant negative TGFbR2 (dnTGFbR 2).
Figures 21A-21C show exemplary results of HT55 liver metastasis models treated with armored anti-GCC CAR T cells.
Figure 22A shows CAR-T cells counted by flow cytometry at designated time points and FACS phenotyping performed. Figure 22B shows percent cytotoxicity in anti-Msln CAR-T cells co-expressing a CAR against Msln with a tgfβ modulator (e.g., tgfβr2-VH or dnTGFbR 2) or GFP (Msln-control VH) with a control VH.
Definition of the definition
In order to make the invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and/or steps of the type described herein, and/or as would become apparent to one of skill in the art upon reading this disclosure, and the like.
And (3) application: as used herein, "administering" a composition to a subject refers to providing, administering, or contacting the composition with the subject. Administration may be accomplished by any of a variety of routes, such as topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.
Adoptive cell therapy: as used interchangeably herein, the term "adoptive cell therapy" or "adoptive cell transfer" or "cell therapy" or "ACT" refers to the transfer of cells (e.g., the genetically modified cell population described herein) into a patient in need thereof. The cells may be derived and propagated from a patient in need thereof (i.e., autologous cells), or may be obtained from a non-patient donor (i.e., allogeneic cells). In some embodiments, the cell is an immune cell, e.g., a lymphocyte, modified to express a CAR and a tgfβ signaling pathway modulator, as described herein (e.g., a tgfβ -armored CAR-T cell). ACT can be performed using a variety of cell types including, but not limited to, natural Killer (NK) cells, T cells, cd8+ cells, cd4+ cells, γδ T cells, regulatory T cells, induced pluripotent stem cells (ipscs), iPSC-derived T cells, iPSC-derived NK cells, hematopoietic Stem Cells (HSCs), mesenchymal Stem Cells (MSCs), and peripheral blood mononuclear cells.
Affinity: as used herein, the term "affinity" refers to the nature of the binding interaction between a binding moiety (e.g., an antigen binding agent (e.g., a variable domain as described herein) and a target (e.g., an antigen (e.g., TGF beta or TGFBR)) and indicates the strength of the binding interactionIn some embodiments, the affinity is measured as a dissociation constant (K D ) And (3) representing. The binding affinity of an antigen binding protein to its target can be determined by equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA)), kinetics (e.g., BIACORE) TM Analysis) or other methods known in the art.
Affinity: as used herein, the term "avidity" is the sum of the intensities of two molecules binding to each other at multiple sites (e.g., taking into account the valency of the interaction).
Animals: as used herein, the term "animal" refers to any member of the kingdom animalia. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and/or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and/or a clone.
And (3) autologous: as used herein, the term "autologous" refers to any material derived from the same individual into which it is later reintroduced.
Allograft: as used herein, "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, the two or more individuals are said to be allogeneic to each other. In some aspects, allogeneic material from individuals of the same species may differ in gene enough for antigen interactions to occur.
Antibodies or antigen binding agents: as used herein, the term "antibody" or "antigen binding agent" refers to a polypeptide that includes typical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. Those skilled in the art will appreciate that the terms mayAre used interchangeably herein. In some embodiments, the term "antibody" or "antigen binding agent" as used herein also refers to an "antibody fragment" or "multiple antibody fragments" that comprise a portion of an intact antibody (e.g., an antigen binding or variable region of an antibody). Examples of "antibody fragments" include Fab, fab ', F (ab') 2 and Fv fragments; a tri-antibody; a four-antibody; a linear antibody; a single chain antibody molecule; and CDR-containing portions included in multispecific antibodies formed from antibody fragments. It will be appreciated by those skilled in the art that the term "antibody fragment" does not imply and is not limited to any particular mode of production. Antibody fragments may be prepared by using any suitable method, including but not limited to cleavage of intact antibodies, chemical synthesis, recombinant production, and the like. As known in the art, naturally occurring intact antibodies are tetrameric agents of about 150kD comprising two identical heavy chain polypeptides (about 50kD each) and two identical light chain polypeptides (about 25kD each) bound to each other to form a so-called "Y-shaped" structure. Each heavy chain comprises at least four domains (each about 110 amino acids long), i.e., amino terminal variable (V H ) Domain (at the top of the Y structure), followed by three constant domains: c (C) H 1、C H 2 and carboxyl terminal C H 3 (at the bottom of the Y-trunk). A short region (referred to as a "switch") connects the heavy chain variable and constant regions. "hinge" will C H 2 and C H The 3 domain is linked to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides in the intact antibody to each other. Each light chain comprises two domains, the amino-terminal variable (V L ) Domain followed by carboxy-terminal constant (C L ) Domains, separated from each other by another "switch". The intact antibody tetramer is composed of two heavy chain-light chain dimers, wherein the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, such that the dimers are connected to each other and form a tetramer. Naturally occurring antibodies are also glycosylated, typically at C H 2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" that is defined by two beta flaps (e.g., 3-,4-or 5-chain flaps) stacked on top of each other in compressed antiparallel beta barrels. Each variable domain contains three hypervariable loops, termed "complementarity determining regions" (CDR 1, CDR2, and CDR 3) and four somewhat invariant "framework" regions (FR 1, FR2, FR3, and FR 4). When the natural antibody is folded, the FR regions form beta flaps that provide the structural framework for the domain, and the CDR loop regions from both the heavy and light chains are clustered together in three dimensions so that they create a single hypervariable antigen binding site at the top of the Y structure. Amino acid sequence comparisons between antibody polypeptide chains have defined two light chain (kappa and lambda) classes, several heavy chain (e.g., mu, gamma, alpha, epsilon, delta) classes, and certain heavy chain subclasses (alpha 1, alpha 2, gamma 1, gamma 2, gamma 3, and gamma 4). Antibody class (IgA [ including IgA1, igA 2) ]IgD, igE, igG [ including IgG1, igG2, igG3 and IgG4 ]]And IgM) are defined based on the class of heavy chain sequences used.
For the purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex that includes sufficient immunoglobulin domain sequence found in a natural antibody, whether such polypeptide is naturally occurring (e.g., produced by an organism reacting an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods, may be referred to and/or used as an "antibody" or "antigen binding agent. In some embodiments, the antibody is a monoclonal antibody; in some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody has a constant region sequence characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody" or "antigen binding agent" as used herein will be understood to encompass (unless otherwise indicated or apparent from context) in appropriate embodiments any construct or form known or developed in the art for capturing antibody structural and functional characteristics in alternative presentation. For example, in some embodiments, the term may refer to bispecific or other multispecific (e.g., enzyme parents, etc.) antibodies, small modular immunopharmaceuticals ("SMIPs" TM ") and single chainAntibodies, camelized antibodies and/or antibody fragments. In some embodiments, the antibody may lack covalent modifications (e.g., linked glycans) that it may have when naturally occurring. In some embodiments, antibodies may contain covalent modifications (e.g., linked glycans, payloads [ e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.)]Or other side groups [ e.g. polyethylene glycol, etc. ]])。
About or about: as used herein, the term "about" or "approximately" as applied to one or more numerical values of interest refers to numerical values similar to the stated reference values. In certain embodiments, unless otherwise specified or apparent from context, the term "about" or "approximately" means within a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) the stated reference value (except where such value would exceed 100% of the possible value). It will be understood that when the term "about" or "approximately" is used to modify a stated reference, it is intended to cover the stated reference as such as well as values that are on either side of the stated reference that are close to the stated reference.
Armored CAR-T cells: as used herein, the term "armored CAR cell" or "armored CAR-T cell" refers to a genetically engineered cell that has the ability to avoid tumor immunosuppression and tumor-induced low CAR-T function. In some embodiments, the armored CAR T cells comprise Chimeric Antigen Receptors (CARs) that recognize cancer-associated antigens and tgfβ signaling pathway modulators.
Complementarity Determining Regions (CDRs): the "CDRs" of the variable domains are amino acid residues within the variable region identified according to the definition of Kabat, chothia, the accumulation of both Kabat and Chothia, abM, contact and/or conformational definitions, or any CDR assay methods well known in the art. The antibody CDRs may be identified as hypervariable regions initially defined by Kabat et al. See, e.g., kabat et al, 1992,Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, NIH, washington d.c. CDR positions alsoCan be identified as the structural loop structure originally described by Chothia and others. See, e.g., chothia et al, nature 342:877-883,1989. Other CDR identification methods include "AbM definition", which is a compromise between Kabat and Chothia and is an AbM antibody simulation software using Oxford Molecular (now ) "contact definition" of CDRs derived from, or based on, observed antigen contacts is as set forth in MacCallum et al, J.mol.biol.,262:732-745,1996. In another approach, referred to herein as "conformational definition" of CDRs, the positions of the CDRs can be identified as residues that contribute enthalpy to antigen binding. See, e.g., makabe et al, journal of Biological Chemistry,283:1156-1166,2008. Still other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDRs, although they may shorten or lengthen depending on the prediction or experimental results of a particular residue or group of residues, and even the entire CDR will not significantly affect antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR may be defined in accordance with any of Kabat, chothia, extension, abM, contact, and/or conformational definitions.
Antibody-dependent cell-mediated cytotoxicity or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells, e.g., natural Killer (NK) cells, neutrophils, and macrophages, enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The antibody "arms" the cytotoxic cells and is necessary to kill the target cells by this mechanism. The primary cells mediating ADCC are NK cells, which express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. Fc expression on hematopoietic cells is summarized in Table 3 at page 464 of Ravetch and Kinet, annu. Rev. Immunol.9:457-92 (1991). In order to assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, for example as described in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al, PNAS USA 95:652-656 (1998).
Antigen: as used herein, the term "antigen" refers to an agent that elicits an immune response; and/or agents that bind to T cell receptors (e.g., when presented by MHC molecules) or antibodies (e.g., produced by B cells) when exposed or administered to an organism. In some embodiments, the antigen elicits a humoral response in the organism (e.g., including the production of antigen-specific antibodies); alternatively or additionally, in some embodiments, the antigen elicits a cellular response in an organism (e.g., a T cell that involves specific interaction of its receptor with the antigen). Those skilled in the art will appreciate that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mouse, rabbit, primate, human) but not in all members of the target organism species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of the target organism species. In some embodiments, the antigen binds to an antibody and/or T cell receptor, and may or may not elicit a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and/or T cell receptor in vitro, whether or not this interaction occurs in vivo. In some embodiments, the antigen reacts with a specific humoral or cellular immune product, including those elicited by a heterologous immunogen.
And (3) associating: as the term is used herein, two events or entities are "associated with" each other if one is related to the presence, level, and/or form of the other. For example, a particular entity (e.g., a polypeptide) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and/or form is correlated with the incidence and/or susceptibility of the particular disease, disorder, or condition (e.g., in a correlated population). In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are and remain in physical proximity to each other. In some embodiments, two or more entities physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are associated in a non-covalent form, such as by means of hydrogen bonding, van der waals interactions (van der Waals interaction), hydrophobic interactions, magnetic properties, and combinations thereof. In some embodiments, when referring to "an antigen associated with a cancer cell," the term "associated with … …" refers to the presence of a particular antigen on the surface of the cancer cell.
Combining: it should be understood that the term "binding" as used herein generally refers to non-covalent association between two or more entities. "direct" bonding involves physical contact between entities or parts; indirect bonding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed under any of a variety of circumstances, including isolated or in the case of more complex systems (e.g., covalently or otherwise associated with a carrier entity and/or in a biological system or cell) entities or portions of an interaction. As used herein, "K a "refers to the rate at which a particular binding moiety associates with a target to form a binding moiety/target complex. As used herein, "K d "refers to the rate of dissociation of a particular binding moiety/target complex. As used herein, "K D "means dissociation constant, which is derived from K d And K is equal to a Ratio (i.e. K) d /K a ) And expressed in molar concentration (M). K (K) D The values may be as known in the artEstablished methods (e.g., by using surface plasmon resonance) or using a biosensor system (e.g.System) to determine.
And (3) a carrier: as used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, the carrier is or includes one or more solid components.
Characteristic parts: as used herein, the term "characteristic portion" is used in its broadest sense to mean that the presence (or absence) of a portion of a substance is associated with the presence (or absence) of a particular feature, attribute, or activity. In some embodiments, a characteristic portion of a substance is a portion of the substance and related substances found to share a particular feature, attribute, or activity, rather than not sharing a particular feature, attribute, or activity.
Chimeric antigen receptor: as used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor composed of one or more of an extracellular target binding domain (e.g., derived from an antibody), a transmembrane region, and one or more intracellular effector domains. CARs are typically introduced into immune cells, such as T cells, to redirect the specificity of a desired cell surface antigen or MHC-peptide complex. These synthetic receptors typically contain a target binding domain that is associated with one or more signaling domains via a flexible linker in a single fusion molecule. The target binding domain is used to direct immune cells (e.g., T cells) to a specific target on the surface of pathological cells (e.g., cancer cells), and the signaling domain contains molecular mechanisms for immune cell (e.g., T cells) activation and proliferation. The flexible linker (i.e., forming a transmembrane domain) that typically passes through the membrane of an immune cell (e.g., T cell) allows the cell membrane to display the target binding domain of the CAR. CARs have successfully redirected immune cells (T cells) against antigens expressed at the surface of tumor cells from various malignant diseases, including lymphomas and solid tumors (Gross et al, (1989) transfer proc.,21 (1 pt 1): 127-30; jena et al, (2010) Blood,116 (7): 1035-44). The extracellular binding domain of the CAR may be composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light chain regions of a murine or humanized monoclonal antibody. In some embodiments, the extracellular binding domain comprises a single domain antibody. Alternatively, scfvs derived from Fab (rather than derived from antibodies obtained, for example, from a Fab library) may be used. In various embodiments, this scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain.
At least three generations of CARs have been developed. The first generation of CARs contained a target binding domain attached to a signaling domain derived from the cytoplasmic region of the cd3ζ or Fc receptor γ chain. First generation CARs have been shown to successfully redirect T cells to selected targets, but they fail to provide long-term expansion and anti-tumor activity in vivo. Second and third generation CARs focus on enhancing modified T cell survival and increasing proliferation by including co-stimulatory molecules such as CD28, OX-40 (CD 134) and 4-1BB (CD 137). Embodiments described herein are directed, in part, to further improving CAR-T-containing immunotherapy, for example, by armoring CAR-T with a tgfβ signaling pathway modulator, thereby making immunotherapy more effective in treating cancer (particularly solid tumor cancer). The armored CARs provided herein may improve or enhance CAR-T function and survival in the face of adverse tumor microenvironment relative to unarmored CAR-T cells.
Codon optimization: as used herein, a "codon optimized" nucleic acid sequence refers to a nucleic acid sequence that has been altered such that translation of the nucleic acid sequence and expression of the resulting protein achieve improved optimization for a particular expression system. The "codon optimized" nucleic acid sequence encodes the same protein as the non-optimized parent sequence on which the "codon optimized" nucleic acid sequence is based. For example, the nucleic acid sequence may be "codon optimized" for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells, etc.), bacterial cells (e.g., e.coli), insect cells, yeast cells, or plant cells.
The comparison can be made: as used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may be different from each other but sufficiently similar to allow comparison therebetween so that a conclusion may be reasonably drawn from observed differences or similarities. Those of ordinary skill in the art will understand what degree of identity is required in any given instance to treat two or more such agents, entities, situations, sets of conditions, etc. as being comparable.
Corresponding to: as used herein, the term "corresponding to" is generally used to designate the position/identity of amino acid residues of a polypeptide of interest. It will be appreciated by those of ordinary skill in the art that for brevity, residues in a polypeptide are typically named using a canonical numbering system based on the reference to the relevant polypeptide, and thus, an amino acid "corresponding to" position 190 residue, for example, does not necessarily actually be the 190 th amino acid in a particular amino acid chain, but corresponds to the 190 th residue in the reference polypeptide; one of ordinary skill in the art will readily understand how to identify "corresponding" amino acids.
Derived from: as used herein, the phrase "derived from" or "specific for a specified sequence" refers to a sequence comprising about at least 6 nucleotides or at least 2 amino acids, at least about 9 nucleotides or at least 3 amino acids, at least about 10-12 nucleotides or 4 amino acids, or at least about 15-21 nucleotides or 5-7 amino acids corresponding to (i.e., identical to or complementary to) a contiguous region of the specified sequence, for example. In certain embodiments, the sequence comprises all specified nucleotide or amino acid sequences. The sequence may be complementary (in the case of polynucleotide sequences) or identical to a sequence region unique to the particular sequence, as determined by techniques known in the art. The regions from which sequences can be derived include, but are not limited to: a region encoding a specific epitope, a region encoding a CDR, a region encoding a framework sequence, a region encoding a constant domain region, a region encoding a variable domain region, and an untranslated and/or nontranscribed region. The derivative sequence is not necessarily physically derived from the sequence of interest under investigation, but may be generated in any manner, including but not limited to chemical synthesis, replication, reverse transcription or transcription, based on information provided by the base sequence in the region from which the polynucleotide is derived. Thus, it may represent a synonymous or antisense orientation of the original polynucleotide. Furthermore, combinations of regions corresponding to a given sequence may be modified or combined in a manner known in the art to suit the intended use. For example, a sequence may comprise two or more consecutive sequences, each comprising a portion of a specified sequence, and interrupted by a region that is different from the specified sequence but is intended to represent a sequence derived from the specified sequence. With respect to antibody molecules, "derived from" includes antibody molecules that are functionally or structurally related to the comparison antibody, e.g., are "derived from" includes antibody molecules having similar or substantially identical sequences or structures, e.g., having identical or similar CDRs, frames, or variable regions. "derived from" an antibody also includes residues, e.g., one or more, e.g., 2, 3, 4, 5, 6 or more residues, which may be contiguous or discontinuous, but are defined or identified according to numbering scheme or homology to the general antibody structure or three-dimensional proximity (i.e., within a CDR or framework region) of the comparison sequence. The term "derived from" is not limited to physical derivatization, but includes the generation by any means, such as by using sequence information from a comparison antibody to design another antibody.
And (3) measuring: many of the methods described herein include an "assay" step. Those of ordinary skill in the art who review this specification will appreciate that this "determination" can be made using any of a variety of techniques available to those of ordinary skill in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, the assay involves manipulation of a physical sample. In some embodiments, the determination involves consideration and/or manipulation of data or information, for example, using a computer or other processing unit adapted to perform a correlation analysis. In some embodiments, the determining involves receiving relevant information and/or material from a source. In some embodiments, the assay involves comparing one or more characteristics of the sample or entity to a comparable reference.
Engineering: as used herein, the term "engineered" describes a polynucleotide, polypeptide, or cell that has been engineered or modified by man and/or its presence and production requiring man-made intervention and/or activity. For example, it is intended to design engineered cells for eliciting a specific effect and that differ from the effect of naturally occurring cells of the same type. In some embodiments, the engineered cells express a chimeric antigen receptor described herein.
Effect function: as used herein, the term "effector function" refers to biological activity attributable to the antigen binding agents described herein. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors; and B cell activation). By "reducing or minimizing" antibody effector function is meant that it is reduced by at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) compared to a wild-type or unmodified antibody. Assays for antibody effector function can be readily determined and measured by one of ordinary skill in the art. In some embodiments, complement fixation, complement dependent cytotoxicity, and antibody dependent cytotoxicity are affected by antibody effector function. In some embodiments, effector function is eliminated via mutations that eliminate glycosylation in the constant region, e.g., "no effector mutations". In one aspect, the null stress mutation is an N297A or DANA mutation in the CH2 region (d265 a+n297A). Shields et al J.biol. Chem.276 (9): 6591-6604 (2001). Alternatively, additional mutations that lead to reduced or eliminated effector function include: K322A and L234A/L235A (LALA). Alternatively, effector function may be reduced or eliminated by production techniques, such as expression in a host cell without glycosylation (e.g., E.coli), or where the glycosylation pattern is caused to change to be ineffective or less effective in promoting effector function (e.g., shinkawa et al, J. Biol. Chem.278 (5): 3466-3473 (2003)).
Epitope: as used herein, the term "epitope" includes any portion that is specifically recognized in whole or in part by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, the epitope consists of multiple amino acids in the antigen. In some embodiments, such amino acid residues are exposed to the surface when the antigen adopts a related three-dimensional conformation. In some embodiments, when the antigen adopts such a conformation, the amino acid residues are physically close or equidistant to each other in space. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearization; e.g., a nonlinear epitope), at least some of the amino acids are physically separated from each other.
Excipient: as used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, e.g., to provide or aid in a desired consistency or stabilization. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
Expression: the term "express" or "expressed," when used in reference to a nucleic acid herein, refers to one or more of the following events: (1) RNA transcript production of the DNA template (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by splicing, editing, 5 'cap formation, and/or 3' end formation); (3) translation of the RNA into a polypeptide; and/or (4) post-translational modification of the polypeptide.
Ex vivo: as used herein, the term "ex vivo" means the process by which cells are removed from a living organism and propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor).
Fusion protein: as used herein, the term "fusion protein" refers to a protein encoded by a nucleic acid sequence engineered from a nucleic acid sequence encoding at least a portion of two different (e.g., heterologous) proteins. The skilled artisan will certainly appreciate that to produce a fusion protein, the nucleic acid sequences are ligated such that the resulting reading frame does not contain internal stop codons.
And (3) a host: the term "host" is used herein to refer to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, non-human primate) or system (e.g., cell or cell line). In some embodiments, the host is an organism to be administered a cell or population of cells expressing a CAR and/or tgfβ modulator described herein. In some embodiments, administering the population of cells results in an improvement in an immune response in the host.
Host cell: as used herein, the phrase "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, the host cell can be used to produce a modified CAR molecule as described herein by standard recombinant techniques. The skilled artisan will appreciate upon reading this disclosure that such terms refer not only to the cells of a particular subject, but also to the offspring of such cells. Since certain modifications may occur in progeny due to mutation or environmental effects, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.
In some embodiments, the host cell is a human cell. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of E.coli, bacillus, streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae, schizosaccharomyces pombe (S.pombe), pichia pastoris (P.pastoris), pichia methanolica (P.methyotica), etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, trichoplusia ni (Trichoplusia ni), etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or tetrad hybridomas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following: CHO (e.g., CHO K1, DXB-11CHO, veggie-CHO), COS (e.g., COS-7), retinal cells, vero, CV1, kidney (e.g., HEK293T, 293EBNA, MSR 293, MDCK, haK, BHK), heLa, hepG2, WI38, MRC 5, colo205, HB 8065, HL-60 (e.g., BHK 21), jurkat, daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, support cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells and cell lines derived from the foregoing. In some embodiments, the cells comprise one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6 TM Cells).
Immune response: as used herein, the term "immune response" refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages or polymorphonuclear cells, to a stimulus such as an antigen or vaccine. An immune response may include any body cell involved in a host defensive response, including, for example, epithelial cells that secrete interferon or cytokines. Immune responses include, but are not limited to, innate and/or adaptive immune responses. Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (e.g., B or T cells), secretion of cytokines or chemokines, inflammation, antibody production, and the like. In some embodiments, the immune response refers to an immune response observed after administration of an armored CAR-T cell or an unarmored CAR-T cell described herein. In some embodiments, the immune response observed after administration of an armored CAR-T cell described herein is measured by one or more of the following: increased proliferation of CAR-expressing cells, increased IFNg production of CAR-expressing cells, increased IL-2 production of CAR-expressing cells, increased proliferation of host immune cells, increased IL-2 production of host immune cells, increased antigen presentation by host antigen presenting cells, increased co-stimulation of host antigen presenting cells, increased activation of endothelial cells, or increased tumor homing of immune cells (e.g., NK cells, T cells, macrophages).
In vitro: as used herein, the term "in vitro" refers to events occurring in an artificial environment (e.g., in a tube or reaction vessel), in a cell culture, etc., rather than in a multicellular organism.
In vivo: as used herein, the term "in vivo" refers to events occurring within multicellular organisms such as humans and non-human animals. In the case of a cell-based system, the term may be used to refer to the occurrence of an event within a living cell (as opposed to, for example, an in vitro system).
Separating: as used herein, the term "isolated" refers to a substance and/or entity that (1) is separated from at least some components associated with the original production (whether in nature and/or in an experimental setting), and/or (2) is designed, produced, prepared, and/or manufactured with human intervention. The isolated substance and/or entity may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% separated from the other components with which it was originally associated. In some embodiments, the purity of the isolated reagent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. As used herein, a substance is "pure" if the substance is substantially free of other components. In some embodiments, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), as will be appreciated by those skilled in the art; in such embodiments, the percent separation or purity of the material is calculated without including such carriers or excipients. As just one example, in some embodiments, a biopolymer, such as a polypeptide or polynucleotide, that is present in nature is considered "isolated" when: a) As its origin or derived source is not associated with some or all of the components that accompany it in its natural state; b) Which is substantially free of other polypeptides or nucleic acids from the same species from which it is naturally produced; c) Expressed by or associated with a component in a cell or other expression system not from the species from which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system that differs from that in which it is naturally produced is considered an "isolated" polypeptide. In some embodiments, the cells may be "isolated cells" isolated from molecules and/or cellular components that naturally accompany the cells. Alternatively or additionally, in some embodiments, cells that have undergone one or more purification techniques are, after reaching the cell that has been associated with: a) Associated with it in nature; and/or b) the extent to which other components associated therewith are separated when initially produced, can be considered "isolated" cells.
And (3) joint: as used herein, the term "linker" refers to a functional group (e.g., a chemical agent or polypeptide) that covalently attaches two or more polypeptides or nucleic acids to each other. As used herein, "peptide linker" refers to one or more amino acids used to couple two proteins together (e.g., to couple VH and VL domains).
Regulator or modulator: as used herein, the term "modulate" or "modulator" refers to the ability of a component to positively or negatively alter the function of interest. Exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%. For example, provided herein are TGFB signaling modulators capable of altering or preventing signaling of a tgfβ receptor. It will be appreciated by those skilled in the art that this may be achieved by binding to a cytokine that activates tgfβr signalling (i.e. tgfβ), or to its receptor itself (e.g. a tgfβantibody or fragment thereof, a TGFBR antibody or fragment thereof). Thus, this term encompasses both molecules that bind tgfβ and molecules that bind tgfβr. In one embodiment, modulators of the present disclosure may neutralize tgfβ signaling through tgfβrii. "neutralizing" means blocking the normal signaling of tgfβ such that the presence of tgfβ has a neutral effect on tgfβrii signaling. In some embodiments, the tgfβ modulator improves an immune response in the host.
Nucleic acid: as used herein, the phrase "nucleic acid" in its broadest sense refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is an oligonucleotide chain or a compound and/or substance that can be incorporated into an oligonucleotide chain via phosphodiester linkages. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide strand comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids" that are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds, as is considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5' -N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of: one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of: one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-adenosine, O (6) -methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to those in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) of polymerization based on complementary templates, propagation in recombinant cells or systems, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the nucleic acid is single stranded; in some embodiments, the nucleic acid is double stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.
Pharmaceutically acceptable vehicle: pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Compositions and formulations suitable for drug delivery of one or more therapeutic compositions are described in Remington's Pharmaceutical Sciences, e.w. martin, mack Publishing co., easton, PA, 15 th edition (1975). In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids which include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol and the like as vehicles. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the bio-neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Polypeptide: a "polypeptide" is generally a string of at least two amino acids linked to each other by peptide bonds. In some embodiments, the polypeptide may comprise at least 3-5 amino acids, each amino acid being linked to other amino acids by at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include "unnatural" amino acids or other entities, which nonetheless can optionally be incorporated into polypeptide chains. In some embodiments, the term "polypeptide" is used to refer to a particular functional class of polypeptides, such as antibodies, chimeric antigen receptors, or co-stimulatory domain polypeptides, and the like. For each such class, the present description provides and/or is known in the art within the class of known amino acid sequences of exemplary polypeptides of several examples; in some embodiments, one or more such known polypeptides are reference polypeptides of the class. In such embodiments, the term "polypeptide" refers to any member of a class that exhibits sufficient sequence homology or identity to a related reference polypeptide that one of ordinary skill in the art would understand to be included in. In many embodiments, members of the representative class also share significant activity with the reference polypeptide. For example, in some embodiments, a member polypeptide exhibits a degree of overall sequence homology or identity of at least about 30-40% with a reference polypeptide, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or comprises at least one region (i.e., a conserved region, typically comprising a characteristic sequence element), which exhibits very high sequence identity, typically greater than 90%, even 95%, 96%, 97%, 98% or 99%. Such conserved regions typically cover at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids.
It will be appreciated that the antibodies and antigen binding agents of the invention may have additional conservative or non-essential amino acid substitutions that have no substantial effect on polypeptide function. Whether a particular substitution can be tolerated, i.e., does not adversely affect the desired biological properties (e.g., binding activity), can be determined as described in Bowie, J U et al, science 247:1306-1310 (1990) or Padlan et al, FASEB J.9:133-139 (1995). A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Prevention of: as used herein, the term "preventing" refers to preventing, avoiding the manifestation of a disease, delaying the onset of, and/or reducing the frequency and/or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., cancer). In some embodiments, prevention is assessed on a population basis, and the agent is considered to "prevent" a disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition.
Pure: as used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a formulation comprising more than about 90% of a particular agent or entity is generally considered to be a pure formulation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
Recombination: as used herein, the term "recombinant" is intended to refer to a polypeptide (e.g., a polypeptide as described herein) that has been designed, engineered, prepared, expressed, created, or isolated by recombinant means, such as a polypeptide expressed by transfection into a host cell using a recombinant expression vector, a polypeptide isolated from a library of recombinant, combinatorial polypeptides, or a polypeptide prepared, expressed, created, or isolated by any other means that involves splicing selected sequence elements into one another. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements and/or combinations thereof are computer-designed. In some embodiments, one or more such selected sequence elements are generated from a combination of a plurality (e.g., two or more) of known sequence elements that are not naturally present in the same polypeptide.
Reference: the term "reference" is often used herein to describe a standard or control agent, individual, population, sample, sequence, or value that is compared to the agent, individual, population, sample, sequence, or value of interest. In some embodiments, the testing and/or assaying of a reference agent, individual, population, sample, sequence, or value is performed substantially simultaneously with the testing or assaying of the agent, individual, population, sample, sequence, or value of interest. In some embodiments, the reference agent, individual, population, sample, sequence, or value is an empirical reference, optionally implemented in a tangible medium. In general, as will be understood by those of skill in the art, a reference agent, individual, population, sample, sequence, or value is determined or characterized under conditions similar to those used to determine or characterize the agent, individual, population, sample, sequence, or value of interest.
Single domain antibodies: as used herein, the term "single domain antibody (sdAb)", "variable single domain" or "immunoglobulin single variable domain (ISV)", "single heavy chain variable domain (VH) antibody" refers to a single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. An sdAb is a single antigen-binding polypeptide with three Complementarity Determining Regions (CDRs). Only sdabs are able to bind antigen without pairing with the corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelized hcabs and their heavy chain variable domains are referred to as "VHHs". Certain VHHs may also be referred to as nanobodies. Camelized sdabs are among the smallest known antigen-binding antibody fragments (see, e.g., hamers-Casterman et al, nature 363:446-8 (1993); greenberg et al, nature 374:168-73 (1995); hassazadeh-Ghasssaboeh et al, nanomedicine (Lond), 8:1013-26 (2013)). The basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3. The camelized VHH domain may be humanised according to standard techniques available in the art and such domains are considered to be "domain antibodies". As used herein, VH includes a camelized VHH domain, and the term VHH thereof may be used to refer to a domain antibody of human or camelized origin comprising only heavy chains. As explained below, some embodiments of aspects of the invention relate to binding agents comprising a single heavy chain variable domain antibody/immunoglobulin heavy chain single variable domain that can bind to tgfβ antigens in the absence of light chains.
The subject: as used herein, the term "subject" refers to any mammal, including humans. In certain embodiments of the invention, the subject is an adult, adolescent or infant. In some embodiments, the term "individual" or "patient" is used and is intended to be interchangeable with "subject. The invention also encompasses administration of the pharmaceutical composition and/or performance of a method of intrauterine treatment. For example, the subject may be a patient suffering from cancer (e.g., of gastrointestinal origin), a symptom of cancer (e.g., a human patient or an animal patient), wherein at least some of the cells express tgfβ, or a patient predisposed to cancer, wherein at least some of the cells express tgfβ. Unless otherwise indicated, the term "non-human animal" according to the present invention includes all non-human vertebrates, such as non-human mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.
Basically: as used herein, the term "substantially" refers to a qualitative condition having all or nearly all of the range or degree of a feature or characteristic of interest. Those of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, accomplish and/or continue to accomplish or achieve or avoid absolute results. Thus, the term "substantially" is used herein to encompass the completeness of the potential lack inherent in many biological and chemical phenomena.
Therapeutic agent: as used herein, the term "therapeutic agent" refers to an agent (e.g., an antigen binding agent) that has biological activity. The term is used herein to refer to a compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material. In some embodiments, the therapeutic agent may be an anticancer agent or a chemotherapeutic agent. As used herein, the term "anti-cancer agent" or "chemotherapeutic agent" refers to an agent having the functional property of inhibiting the development or progression of a human tumor, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis or angiogenesis is often a property of anticancer or chemotherapeutic agents. The chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation.
Conversion: as used herein, refers to any process of introducing exogenous DNA into a host cell. Transformation can be performed under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, the particular transformation method is selected based on the host cell being transformed, and may include, but is not limited to: viral infection, electroporation, mating, lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replication as an autonomously replicating plasmid or as part of a host chromosome. In some embodiments, the transformed cells transiently express the introduced nucleic acid for a limited period of time.
Transforming growth factor-beta (tgfβ): as used herein, the terms "TGF- β", "TGFb", "tgfβ" and "transforming growth factor- β" are used interchangeably herein and refer to a family of molecules having the full length natural amino acid sequence of any TGF- β from a human, including their latent forms as well as the binding or unbound complexes of precursors with mature TGF- β ("latent TGF- β"). Such TGF- β mentioned herein will be understood to refer to any of the presently identified forms, including TGF- β1, TGF- β2, TGF- β3, TGF- β4 and TGF- β5 and their potential forms, as well as human TGF- β species identified in the future, including polypeptides derived from any known TGF- β sequence and being at least about 75%, preferably at least about 80%, more preferably at least about 85%, still more preferably at least about 90% and even more preferably at least about 95% homologous to said sequence. Specific terms "TGF- β1", "TGF- β2" and "TGF- β3", and "TGF- β4" and "TGF- β5" refer to TGF- βs defined in the literature, such as Derynck et al, nature, supra; seyedin et al, j.biol.chem.,262, supra; and demaptin et al, supra. The term "TGF- β" refers to a gene encoding human TGF- β. Preferred TGF-beta is a human TGF-beta natural sequence.
Members of the TGF-beta family are defined as those members that have 9 cysteine residues in the mature portion of the molecule, share at least 65% homology with other known TGF-beta sequences in the mature region, and compete for the same receptor. Furthermore, they all appear to encode larger precursors that share a highly homologous region near the N-terminus and that show three cysteine residues remaining in the portion of the precursor that will be later removed by processing. Furthermore, TGF- β appears to have four or five amino acid processing sites.
Transforming growth factor-beta receptor (tgfβr): as used herein, the term "TGF-bR" or "TGF-b receptor" or "TGF- β receptor" or "tgfβr" is used to encompass all three subtypes of the tgfβr family (i.e., tgfβr1, tgfβr2, tgfβr3). Tgfβ receptors are characterized by serine/threonine kinase activity and exist in several different homodimeric isoforms.
Tgfβ signaling pathway modulators or tgfβ modulators: as used herein, the term "tgfβ signaling pathway modulator" or "tgfβ modulator" interchangeably refers to a molecule (e.g., an antibody or fragment thereof) capable of modulating a tgfβ signaling pathway (e.g., having inhibitory, blocking, or neutralizing effects), and which can bind to tgfβ itself or which can bind to a tgfβ receptor on a cell. In either case, the modulator inhibits the tgfβ signaling pathway (e.g., by binding to the cytokine (i.e., tgfβ) itself or by binding to the receptor for tgfβ). Thus, this term encompasses both types of modulators that bind tgfβ and bind to tgfβ receptors. In various embodiments described herein, a tgfβ signaling pathway modulator is expressed in a modified immune cell (e.g., CAR-T cell) along with a chimeric antigen receptor. CAR-T cells expressing such tgfβ signaling pathway modulators are referred to herein as tgfβ -armored CAR-T cells.
Treatment: as used herein, the term "treatment" or "treatment" is defined as administration of a therapeutic agent to a subject, such as a patient, or to a tissue or cell isolated from the subject and returned to the subject (e.g., by application). In some embodiments, the therapeutic agent is an armored CAR-T cell (e.g., an engineered CAR T cell that co-expresses a tgfβ modulator). The treatment may be a cure, alleviation, relief, alteration, remedy, improvement, alleviation, improvement, or a predisposition to affect a disorder, a symptom of a disorder, or a condition (e.g., cancer). While not wishing to be bound by theory, it is believed that treatment may cause inhibition, ablation, or killing of cells in vitro or in vivo, or otherwise reduce the ability of cells (e.g., abnormal cells) to mediate a disorder, such as the disorders described herein (e.g., cancer).
The invention described herein is used in "effective amounts" for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of an armored CAR-T cell described herein (e.g., an engineered cell that coexpresses a CAR and a modulator of tgfβ signaling) is an amount effective to ameliorate or reduce one or more symptoms of a disease or to prevent or cure a disease (e.g., cancer).
Variable region or domain: as used herein, the term "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same class) and contain antigen binding sites. Heavy chain-only antibodies have a single heavy chain variable region.
And (3) a carrier: as used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which additional DNA segments may be ligated. Another vector type is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".
Detailed Description
The present invention provides methods and compositions for enhancing immune responses to cancer using modified immune cells (e.g., CAR-T cells) that are armored with polypeptides that modulate tgfβ signaling. The invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, unless indicated otherwise, since the scope of the present invention will be limited only by the appended claims.
TGF-B/SMAD signaling
Transforming growth factor beta (TGF-beta) is a multifunctional cytokine originally named for its ability to transform normal fibroblasts into cells capable of independent anchorage growth. TGF- β signaling controls many critical cellular functions including proliferation, differentiation, survival, migration, and epithelial-mesenchymal transition. It regulates a variety of biological processes such as extracellular matrix formation, wound healing, embryonic development, skeletal development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Deregulation of TGF- β leads to pathological conditions such as birth defects, cancer, chronic inflammation, autoimmune and fibrotic diseases.
TGF- β is produced primarily by hematopoietic and neoplastic cells, which can regulate (i.e., stimulate or inhibit) the growth and differentiation of cells from a variety of normal and neoplastic tissue sources (Spom et al, science,233:532 (1986)), and stimulate the formation and development of various matrix elements. TGF-beta is involved in many proliferative and non-proliferative cellular processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses.
TGF-beta also possesses immunosuppressive activity, including lymphokine activated killer cell (LAK), cytotoxic T Lymphocyte (CTL) inhibition, reduction of B cell lymphopoiesis and kappa light chain expression, negative regulation of hematopoiesis, down-regulation of HLA-DR expression on tumor cells, and inhibition of antigen activated B lymphocyte proliferation in response to B cell growth factors. Many human tumors and many tumor cell lines produce TGF- β, suggesting a possible mechanism for those tumors to evade normal immune surveillance. In addition to the observation that some transformed cell lines have lost the ability to react in an autocrine manner to TGF- β, which stimulates matrix formation and reduces tumor immune surveillance, this negative immune regulator suggests an attractive model for neoplastic deregulation and proliferation.
Since TGF- β signaling is important for both healthy cells and cancer regulation, systemic targeting of TGF- β can cause unwanted side effects. In particular with respect to cancer, members of the TGF- β family are known to possess many biological activities associated with tumorigenesis (including angiogenesis) and metastasis. TGF-beta inhibits proliferation of many cell types, including capillary endothelial cells and smooth muscle cells. Modulation of synthase expression (α1β1, α2β1, and αvβ3, which are involved in endothelial cell migration) under TGF- β. Integrins are involved in migration of all cells, including metastatic cells. TGF-beta down regulates matrix metalloproteinase expression required for angiogenesis and metastasis. TGF-beta induces inhibitors of plasminogen activator, which inhibit the protease cascade required for angiogenesis and metastasis. TGF-beta induces normal cells to inhibit transformed cells. See, e.g., YIngling et al, nature Reviews,3 (12): 1011-1022 (2004), which discloses the pathogenesis of a variety of diseases, including cancer and fibrosis, and proposes a theoretical basis for evaluation of TGF-beta signaling inhibitors as cancer therapeutics, biomarkers/diagnostics, small molecule inhibitor structures in development, and targeted drug development models for application in their development.
As used herein, the term "TGF- β signaling pathway" is used to describe downstream signaling events due to TGF- β and TGF- β like ligands. For example, in one signaling pathway, TGF-beta ligands bind to and activate type II TGF-beta receptors. Type II TGF- β receptors recruit and form heterodimers with type I TGF- β receptors. The resulting heterodimer allows for phosphorylation of type I receptors, which in turn phosphorylates and activates members of the SMAD protein family. As is well known to those skilled in the art, the signaling cascade is triggered and ultimately leads to control of mediator expression involving cell growth, cell differentiation, tumor formation, apoptosis, cell homeostasis, and the like. It is also contemplated that other TGF- β signaling pathways may operate according to the methods described herein.
Modulators of TGF-beta signaling pathway
The present invention provides an immune modulating system comprising a modulator of TGF-beta signaling (e.g., a polypeptide that modulates TGF-beta signaling or a nucleic acid sequence encoding a polypeptide that modulates TGF-beta signaling). In some embodiments, a modulator of TGF-beta signaling may elicit a cellular response upon binding to TGF-beta or TGF-beta receptor. In some embodiments, the modulator of TGF- β signaling is secreted from the cell.
In various embodiments, the invention provides modified immune cells (e.g., T cells) that together express a chimeric antigen receptor and a modulator of TGF- β signaling. Such modulators may bind to TGF-beta itself or to TGF-beta receptors. CAR-T cells expressing such modulators are referred to herein as TGF-beta armored CAR-T cells.
anti-TGF beta and anti-TGF beta R2 antigen binding molecules
In some embodiments, the modulator of TGF- β signaling is an antigen binding molecule (e.g., an antibody or antigen binding fragment thereof). In some embodiments, the antigen binding molecule (e.g., an antibody or antigen binding fragment thereof) specifically binds to TGF- β. In some embodiments, the antigen binding molecule (e.g., an antibody or antigen binding fragment thereof) specifically binds to a TGF-beta receptor (tgfβr) (e.g., tgfβr1, tgfβr2).
A modulator of TGF-beta signaling (e.g., an anti-tgfβ antibody molecule or an anti-tgfβr antibody molecule) may comprise all or an antigen-binding subset of CDRs or heavy chains as described herein. Exemplary amino acid sequences (including variable regions) of anti-tgfβ or anti-tgfβr2 antigen binding agents described herein are shown in table 1. Other anti-tgfβ or anti-tgfβr2 antibodies are also described in U.S. patent nos. 7,723,486 and 9,783,604; U.S. patent application publication nos. US20160017026A1 and US20180105597, US 20190119387; international patent applications WO2012093125A1, WO 2011/012639, WO 2017/141208 Al; each of which is hereby incorporated by reference in its entirety. Antigen binding agents useful in the immunomodulatory systems described herein include, but are not limited to, antibodies that specifically bind to an antigen (e.g., tgfβr epitope), bivalent fragments such as (Fab') 2, monovalent fragments such as Fab, single chain antibodies, single chain Fv (scFv), single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, and the like.
In some embodiments, the immune modulation system comprises a TGF-beta signaling modulator (e.g., an anti-tgfβ or anti-tgfβr2 antigen binding agent) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences provided in table 1. In some embodiments, the immune modulation system comprises a TGF- β signaling modulator comprising one or more CDR sequences of an antibody or fragment thereof described in table 1. In some embodiments, a TGF-b signaling modulator of the invention comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a VH sequence provided in table 1. In some embodiments, the VH of a modulator of TGF- β signaling is a single domain antibody (VH).
In some embodiments, a modulator of TGF- β signaling comprises a leader sequence. In some embodiments, the TGF- β signaling modulator is a monomer. In some embodiments, the TGF- β signaling modulator is a dimer. In some embodiments, the TGF- β signaling modulator is a trimer.
In some embodiments, the TGF- β signaling modulator comprises a linker of tandem connection domains. In some embodiments, the linker comprises GGGGS (SEQ ID NO: 59). In some embodiments, the linker comprises (GGGGS) n (SEQ ID NO: 59), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 61).
In some embodiments, the TGF- β signaling modulator comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a VL sequence provided in table 1. In some embodiments, the anti-tgfβ antigen binding agents of the present invention comprise the same heavy chain variable region amino acid sequences as the VH sequences provided in table 1.
In some embodiments, a modulator of TGF- β signaling of the invention comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a vH sequence provided in table 1. In some embodiments, a TGF- β signaling modulator of the invention comprises the same heavy chain variable region amino acid sequence as the vH sequence provided in table 1.
In some embodiments, the VH of the TGF- β signaling modulator (e.g., a single domain antibody) comprises a leader sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that provided in table 1. In some embodiments, the vH anti-tgfβ antigen binding agent (e.g., a single domain antibody) comprises a leader sequence provided in table 1.
TABLE 1 exemplary anti-TGF beta and anti-TGF beta R2 antigen binding molecules
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In some embodiments, the anti-tgfβ or anti-tgfβr antigen binding agent is an antibody. Typical building blocks of naturally occurring mammalian antibodies are tetramers. Each tetramer is composed of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains can be divided into kappa and lambda light chains. Heavy chains can be categorized as mu, delta, gamma, alpha or epsilon and define the isotype of antibodies as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See generally chapter 7 of Fundamental Immunology (Paul, W.code, 2 nd edition, raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form an antibody binding site. The preferred isotype of anti-tgfβ antibody molecules is IgG immunoglobulins, which can be divided into four subtypes, igG1, igG2, igG3, and IgG4, each having a different gamma heavy chain. Most therapeutic antibodies are human, chimeric or humanized antibodies of the IgG1 type. In a particular embodiment, the anti-tgfβ antibody molecule has an IgG1 isotype.
The variable regions of each heavy and light chain pair form an antigen binding site. Thus, an intact IgG antibody has two identical binding sites. However, a bifunctional or bispecific antibody is an artificial hybridization construct having two different heavy/light chain pairs, resulting in two different binding sites.
The chains all exhibit the same general structure of relatively conserved Framework Regions (FR) joined by three hypervariable regions (also known as complementarity determining regions or CDRs). CDRs from each pair of two chains are aligned by the framework regions and are able to bind to specific epitopes. From N-terminal to C-terminal, both the light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acids of each domain are specified according to the definition of the following documents: kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)) or Chothia and Lesk J.mol.biol.196:901-917 (1987); chothia et al Nature 342:878-883 (1989). As used herein, CDR refers to each of the heavy chain (HCDR 1, HCDR2, HCDR 3) and the light chain (LCDR 1, LCDR2, LCDR 3).
Thus, in one embodiment, the antibody molecule comprises one or both of the following:
(a) One, two, three or antigen binding numbers of the light chain CDRs (LCDR 1, LCDR2 and/or LCDR 3) of one of the human hybridomas, selected lymphocytes or murine antibodies described above. In embodiments, the CDRs may comprise one or more or all of the amino acid sequences of LCDR1-3 as follows: LCDR1 or modified LCDR1 wherein one to seven amino acids are conservatively substituted; LCDR2 or modified LCDR2 wherein one or both amino acids are conservatively substituted; or LCDR3 or modified LCDR3 wherein one or both amino acids are conservatively substituted; and
(b) One, two, three or antigen binding numbers of heavy chain CDRs (HCDR 1, HCDR2 and/or HCDR 3) of one of the human hybridomas, selected lymphocytes or murine antibodies described above. In embodiments, the CDRs may comprise the amino acid sequences of one or more or all of the following HCDRs 1-3: HCDR1 or a modified HCDR1 wherein one or both amino acids are conservatively substituted; HCDR2 or a modified HCDR2 in which one to four amino acids are conservatively substituted; or HCDR3 or modified HCDR3, wherein one or both amino acids are conservatively substituted.
In some embodiments, an anti-tgfβ antibody molecule or anti-tgfβr (e.g., anti-tgfβr2) antibody molecule of the invention can cause Antibody Dependent Cellular Cytotoxicity (ADCC) of cells expressing tgfβ (e.g., tumor cells). Antibodies with IgG1 and IgG3 isotypes can be used to elicit effector functions of antibody-dependent cellular cytotoxicity due to their ability to bind to Fc receptors. Antibodies with IgG2 and IgG4 isotypes can be used to minimize ADCC reactions because of their low ability to bind Fc receptors. In related embodiments, changes in substitution or glycosylation composition can be made in the antibody Fc region, for example by growth in a modified eukaryotic cell line, to enhance Fc receptor recognition, binding and/or mediating cytotoxicity of anti-TGF-beta antibodies or anti-TGF-beta R (e.g., anti-TGF-beta R2) antibody-bound cells (see, e.g., U.S. Pat. No. 7,317,091, U.S. Pat. No. 5,624,821 and disclosures including WO 00/42072, shields et al, J.biol. Chem.276:6591-6604 (2001), lazar et al, proc. Natl. Acad. Sci. U.S. A.103:4005-4010 (2006), satoh et al, expert Opin biol. Ther.6:1161-1173 (2006)). In certain embodiments, an antibody or antigen-binding fragment (e.g., humanized antibody, human antibody) may comprise amino acid substitutions or substitutions that alter or tailor a function (e.g., effector function). For example, a human-derived constant region (e.g., γ1 constant region, γ2 constant region) can be designed to reduce complement activation and/or Fc receptor binding. (see, e.g., U.S. Pat. No. 5,648,260 (Winter et al), U.S. Pat. No. 5,624,821 (Winter et al), and U.S. Pat. No. 5,834,597 (Tso et al), the entire teachings of which are incorporated herein by reference in their entirety). Preferably, the human constant region amino acid sequence containing such amino acid substitutions or replacements is at least about 95% identical over the entire length to the amino acid sequence of the human constant region that has not been altered, and more preferably at least about 99% identical over the entire length to the amino acid sequence of the human constant region that has not been altered. Additional anti-TGF-beta antigen binding molecules are further described in U.S. Pat. No. 8,785,600 (Nam et al), the entire teachings of which are incorporated herein by reference.
In yet another embodiment, effector function may also be altered by modulating the glycosylation pattern of the antibody. Alterations refer to the deletion of one or more saccharide moieties found in the antibody, and/or the addition of one or more glycosylation sites not present in the antibody. For example, antibodies with enhanced ADCC activity and mature saccharide structures lacking fucose linked to the Fc region of the antibody are described in U.S. patent application publication No. 2003/0157108 (Presta). See also U.S. patent application publication No. 2004/0093621 (Kyowa Hakko Kogyo co., ltd.). Glycofi also developed a yeast cell line capable of producing antibody-specific glycoforms.
Additionally or alternatively, antibodies with altered glycosylation patterns, such as low fucosylation antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNac structure, can be prepared. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such saccharide modification may be achieved by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells having altered glycosylation machinery have been described in the art and can be used as host cells, wherein the recombinant antibodies of the invention are engineered to be expressed to produce antibodies having altered glycosylation. For example, EP 1,176,195 (Hang et al) describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such cell lines exhibit low fucosylation. PCT publication No. WO 03/035835 (Presta) describes a variant CHO cell line, lec13 cells, which have a reduced capacity to link fucose to Asn (297) linked carbohydrates, and also result in antibodies expressed in the host cells exhibiting low fucosylation (see also Shields, R.L. et al, 2002J. Biol. Chem. 277:26733-26740). PCT publication No. WO 99/54342 (Umana et al) describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., beta (1, 4) -N-acetylglucosaminyl transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al, 1999Nat. Biotech.17:176-180).
Humanized antibodies can also be prepared using CDR grafting methods. Techniques for producing such humanized antibodies are known in the art. Typically, humanized antibodies are produced by obtaining nucleic acid sequences encoding the variable heavy and variable light chain sequences of antibodies that bind tgfβ, identifying complementarity determining regions or "CDRs" in the variable heavy and variable light chain sequences, and grafting the CDR nucleic acids onto human framework nucleic acid sequences. (see, e.g., U.S. Pat. nos. 4,816,567 and 5,225,539). The positions of the CDRs and framework residues can be determined (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. device of Health and Human Services, NIH publication No. 91-3242, and Chothia, C. Et al, J.mol. Biol.196:901-917 (1987)).
In some embodiments, the immune modulatory systems of the invention comprise nucleic acid sequences encoding anti-tgfβ or anti-tgfβr antibody molecules from the CDRs of the antibody molecules described in table 1. In some embodiments, the sequences from table 1 may be incorporated into a molecule that recognizes tgfβ or tgfβr for use in the methods of treatment described herein (e.g., immune modulating systems, immunoreactive cells, or methods of treatment comprising the same). The selected human framework is one suitable for in vivo administration, meaning that it does not exhibit immunogenicity. For example, such a determination may be made via previous experience with in vivo use of such antibodies and amino acid similarity studies. Suitable framework regions may be selected from the following human source antibodies: it has at least about 65% amino acid sequence identity, and preferably at least about 70%, 80%, 90% or 95% amino acid sequence identity, over the length of the framework region within the amino acid sequence of the equivalent portion (e.g., framework region) of the donor antibody (e.g., anti-tgfβ antibody molecule). The amino acid sequence identity can be determined using a suitable amino acid sequence alignment algorithm, such as CLUSTAL W, using preset parameters. (Thompson J.D. et al, nucleic Acids Res.22:4673-4680 (1994)).
Once the CDRs and FRs of the cloned antibody to be humanized are identified, the amino acid sequences encoding the CDRs can be identified and the corresponding nucleic acid sequences grafted onto the selected human FRs. This can be done using known primers and adaptors, the selection of which is known in the art. All CDRs of a particular human antibody may be replaced by at least a portion of non-human CDRs, or only some CDRs may be replaced by non-human CDRs. Only the number of CDRs required for binding of the humanized antibody to a predetermined antigen need be replaced. After CDR grafting onto selected human FR, the resulting "humanized" variable heavy and variable light chain sequences are expressed to produce humanized Fv or humanized antibodies that bind to tgfβ or tgfβr. Preferably, the CDR-grafted (e.g., humanized) antibody binds tgfβ or tgfβr with an affinity similar, substantially the same or better than the affinity of the donor antibody. Typically, the humanized variable heavy and light chain sequences are expressed as fusion proteins with human constant domain sequences, thus obtaining an intact antibody that binds tgfβ. However, humanized Fv antibodies may be produced that do not comprise such constant sequences.
Humanized antibodies in which specific amino acids have been substituted, deleted or added are also within the scope of the present invention. In particular, humanized antibodies may have amino acid substitutions in the framework regions, for example, to improve binding to an antigen. For example, a selected, small number of acceptor framework residues of a humanized immunoglobulin chain may be replaced with the corresponding donor amino acid. Substitution positions include amino acid residues adjacent to the CDR, or amino acid residues capable of interacting with the CDR (see, e.g., U.S. Pat. No. 5,585,089 or 5,859,205). The recipient framework may be a mature human antibody framework sequence or a consensus sequence. As used herein, the term "consensus sequence" refers to a sequence that is most common or designed from the most common residues at each position of the sequence in a region in a related family member. There are a variety of human antibody consensus sequences that can be used, including consensus sequences of different subsets of the human variable region (see Kabat, E.A. et al Sequences of Proteins of Immunological Interest, fifth edition, U.S. device of Health and Human Services, U.S. device Printing Office (1991)). The Kabat database and its applications are available on-line free, for example via IgBLAST (see also Johnson, g. And Wu, t.t., nucleic Acids Research, 29:205-206 (2001)) of the national center for biotechnology information (National Center for Biotechnology Information, bethesda, md.) of bescens, maryland.
In certain embodiments, the tgfβ or tgfβr antibody molecule is a human anti-tgfβ or anti-tgfβr IgG1 antibody. Since such antibodies have the desired binding to tgfβ or tgfβr molecules, either of such antibodies can be readily isotyped switched to produce human IgG4 isotypes, e.g., while still having the same variable regions (which define the specificity and affinity of the antibody to a degree). Thus, when antibody candidates are generated that meet the desired "structural" attributes as discussed above, they can generally provide at least some of the additional "functional" attributes desired via isotype switching.
Single chain antibody
Single chain antibodies lack some or all of the constant domains of the intact antibody from which they are derived. Thus, they can overcome some of the problems associated with the use of whole antibodies. For example, single chain antibodies tend to be free of certain unwanted interactions between heavy chain constant regions and other biomolecules. In addition, single chain antibodies are significantly smaller than intact antibodies and can have greater permeability than intact antibodies, allowing the single chain antibodies to more efficiently localize and bind to target antigen binding sites. Furthermore, the relatively small size of single chain antibodies compared to whole antibodies makes them less likely to elicit an inappropriate immune response in the recipient.
In some embodiments, the tgfβ signaling modulator is a single chain antigen binding molecule (e.g., scFv) that specifically binds to tgfβ. In some embodiments, the tgfβ signaling modulator is a single chain antigen binding molecule (e.g., scFv) that specifically binds to a TGF-B receptor (tgfβr) (e.g., tgfβr1, tgfβr2).
Multiple single chain antibodies (each single chain having one VH and one VL domain covalently linked by a first peptide linker) may be covalently linked by at least one or more peptide linkers to form multivalent single chain antibodies, and which may be monospecific or multispecific. Each chain of a multivalent single chain antibody comprises a variable light chain fragment and a variable heavy chain fragment, and is linked to at least one other chain by a peptide linker. The peptide linker consists of at least 15 amino acid residues. The maximum number of linker amino acid residues is about 100.
Two single chain antibodies may be combined to form a diabody, also known as a bivalent dimer. Diabodies have two chains and two binding sites, and may have mono-or bispecific properties. Each chain of the diabody comprises a VH domain linked to a VL domain. The domains are linked to a linker that is short enough to prevent pairing between domains on the same strand, thereby facilitating pairing between complementary domains on different strands, thereby regenerating two antigen binding sites.
Three single chain antibodies may be combined to form a trivalent antibody, also known as a trivalent trimer. Trivalent antibodies are composed of a direct fusion of the amino acid terminus of a VL or VH domain with the carboxy terminus of the VL or VH domain (i.e., without any linker sequences). Trivalent antibodies have three Fv heads in which the polypeptides are arranged in a circular, head-to-tail fashion. One possible trivalent antibody conformation is planar, with three binding sites lying in one plane at an angle of 120 degrees to each other. Trivalent antibodies may have mono-, di-or trispecificity.
Single domain antibodies
Single domain antibodies (sdabs) differ from conventional 4-chain antibodies by having a single monomeric antibody variable domain. For example, camelids and sharks produce sdabs called heavy chain-only antibodies (hcabs), which are naturally devoid of light chains. Camelized heavy chain-only antibodies have a single heavy chain variable domain (VHH) for the antigen binding fragment in each arm, which can have high affinity for the antigen without the aid of light chains. The camelized VHH is referred to as the smallest functional antigen binding fragment and has a molecular weight of about 15kD.
One aspect of the application provides an isolated single domain antibody (referred to herein as an "anti-tgfβr sdAb") that specifically binds to tgfβr (e.g., human tgfβr2). In some embodiments, the anti-tgfβr sdAb modulates tgfβ activity. In some embodiments, the anti-tgfβr sdAb is an antagonist antibody. Further provided are antigen binding fragments derived from any of the anti-tgfβr sdabs described herein, and antigen binding proteins comprising any of the anti-tgfβr sdabs described herein. In some embodiments, the anti-tgfβr sdAb comprises one, two, and/or three CDR sequences provided in table 1. Exemplary anti-tgfβr sdabs are provided in table 1.
In some embodiments, some or all of the CDR sequences, heavy chains, may be used in another antigen binding agent, for example in a CDR-grafted, humanized or chimeric antibody molecule. Embodiments include antibody molecules that contain sufficient CDRs (e.g., all three CDRs from one of the heavy chain variable regions described above) to bind tgfβ.
In some embodiments, the CDRs (e.g., all HCDRs) are embedded in a human or human-derived framework region. Examples of human framework regions include human germline (germline) framework sequences, affinity matured (in vivo or in vitro) human germline sequences, or synthetic human sequences, e.g., consensus sequences. In one embodiment, the heavy chain framework is an IgG1 or IgG2 framework.
In some embodiments, the tgfβ modulators of the present invention comprise the heavy chain variable region amino acid sequences provided in table 1. In some embodiments, the anti-tgfβ antigen binding agent is an antibody having only a single domain heavy chain (e.g., an antigen binding agent that does not comprise an immunoglobulin light chain).
Antibody fragments for therapeutic or diagnostic use in vivo may benefit from modifications that enhance their serum half-life. The organic moiety intended for increasing the in vivo serum half-life of the antibody may comprise one, two or more linear or branched moieties selected from the group consisting of: hydrophilic polymer groups (e.g., linear or branched polymers (e.g., polyalkylene glycols such as polyethylene glycol, monomethoxy-polyethylene glycol, and the like), sugars (e.g., dextran, cellulose, polysaccharide, and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyaspartic acid, and the like), polyalkylene oxides, and polyvinylpyrrolidone), fatty acid groups (e.g., monocarboxylic or dicarboxylic acids), fatty acid ester groups, lipid groups (e.g., diacyl glycerol groups, sphingolipid groups (e.g., ceramide groups), or phospholipid groups (e.g., phosphatidylethanolamine groups). Preferably, the organic moiety binds to a predetermined site at which the organic moiety does not impair the function of the resulting immunoconjugate (e.g., reduce antigen binding affinity) compared to the unconjugated antibody moiety. The organic moiety may have a molecular weight of about 500Da to about 50,000Da, preferably about 2000, 5000, 10,000 or 20,000 Da. Examples and methods of modifying polypeptides (e.g., antibodies) with organic moieties can be found, for example, in U.S. Pat. Nos. 4,179,337 and 5,612,460, PCT publication Nos. WO 95/06058 and WO 00/26156, and U.S. patent application publication No. 20030026805.
TGF beta R extracellular domains
The TGF-beta receptor contemplated for use in the immune modulation system described herein may be any TGF-beta receptor, including receptors from the activin-like kinase family (ALK), the Bone Morphogenic Protein (BMP) family, the Nodal family, the growth and differentiation factor family (GDF), and the TGF-beta receptor family. TGF-beta receptors are serine/threonine kinase receptors that affect various growth and differentiation pathways in cells. In some embodiments, the tgfβ signaling modulator is an engineered recombinant extracellular domain (ECD) of a tgfβ receptor (e.g., tgfβr1, tgfβr2). In some embodiments, the TGF- β receptor useful in the immune modulation systems described herein is a type II TGF- β receptor (e.g., TGF- βR2).
In some embodiments, the tgfβ modulator comprises the tgfβr provided in table 2. In some embodiments, the tgfβ modulator comprises a sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence provided in table 2.
TABLE 2 exemplary TGF beta R extracellular Domains
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Chimeric antigen receptor
In some aspects, the invention provides an immune modulation system comprising a modulator of TGF- β signaling (e.g., a polypeptide that modulates TGF- β signaling or a nucleic acid sequence encoding a polypeptide that modulates TGF- β signaling) and a Chimeric Antigen Receptor (CAR) that can bind to an antigen of interest. CAR is a hybrid molecule comprising three basic units: (1) extracellular antigen binding motif, (2) linking/transmembrane motif, and (3) intracellular T cell signaling motif (Long AH, haso W M, orentas R J. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncominmunology.2013; 2 (4): e 23621). In some embodiments, the CARs of the invention comprise a signal or leader peptide, an antigen binding domain, a transmembrane and/or hinge domain, a costimulatory domain, and an intracellular domain from the N-terminus to the C-terminus. In some embodiments, the CAR is a "first generation CAR," e.g., a CAR that includes only a CD3 zeta signal upon antigen binding. "second generation CARs" include CARs that provide co-stimulation (e.g., CD28 or CD 137) and activation (cd3ζ) simultaneously. "third generation CARs" include CARs that provide multiple co-stimulations (e.g., CD28 and CD 137) and activation (CD 3). In various embodiments, the CAR is selected to have a high affinity or avidity for the antigen.
The antigen binding motif of a CAR is typically formed after a single chain fragment variable domain (ScFv), the minimal binding domain of an immunoglobulin (Ig) molecule, or a single domain antibody (e.g., WO2018/028647 A1). Alternative antigen binding motifs, such as receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressing IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG 2D) have also been engineered. Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown C E et al, clin Cancer res.2012;18 (8): 2199-209; lehner M et al, PLoS one.2012;7 (2): E31210).
In some embodiments, the antigen binding domain of the CAR is a single chain variable fragment. In some embodiments, the antigen binding domain of the CAR is a single domain antibody. In some embodiments, the CAR comprises a signal or leader peptide, vH, CD28 transmembrane and hinge, CD28 costimulatory domain, and cd3ζ intracellular domain from N-terminus to C-terminus.
The CAR's linking motif can be a relatively stable domain, such as the constant domain of IgG, or a flexible linker designed to extend. Structural motifs, such as those derived from IgG constant domains, can be used to extend ScFv binding domains away from T cell membrane surfaces. This may be important for certain tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., disialoganglioside GD2; orentas et al, unpublished observations). The signaling motif used in CARs has so far always included a CD3- ζ chain, as this core motif is a key signal for T cell activation. The first reported second generation CAR has a CD28 signaling domain and a CD28 transmembrane sequence. This motif is also used in third generation CARs which contain the CD137 (4-1 BB) signaling motif (Zhao Y et al J Immunol.2009;183 (9): 5563-74). With the advent of new technology, T cells were activated with beads that bound to anti-CD 3 and anti-CD 28 antibodies, and the advent of classical "signal 2" from CD28, no longer needed to be encoded by the CAR itself. Using bead activation, the third generation vectors were found to be not superior to the second generation vectors in vitro assays, and they were not significantly superior to the second generation vectors in leukemia mouse models (Haso W, lee D W, shah N, stetler-Stevenson M, yuan C M, pastan I H, dimitrov D S, morgan R A, fitzGerald D J, barrett D M, wayne A S, mackall C L, orentas R J.anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, blood.2013;121 (7): 1165-74;Kochenderfer J N et al, blood.2012;119 (12): 2709-20). The clinical success of CD 19-specific CAR in second generation CD28/CD 3-zeta (Lee D W et al American Society of Hematology Annual meeting. New Orleans, la.; dec.7-10,2013) and CD137/CD 3-zeta signaling format (Porter D L et al N Engl J Med.2011;365 (8): 725-33) demonstrated this. In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 are also capable of providing important persistence signals in CAR-transduced T cells (Yvon E et al, clin Cancer Res.2009;15 (18): 5852-60). Also important are the culture conditions under which the CAR T cell population is cultured.
Features of the CAR include its ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, and to take advantage of the antigen binding properties of monoclonal antibodies. non-MHC-restricted antigen recognition enables CAR-expressing T cells to recognize antigens that are not involved in antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize exogenous T Cell Receptor (TCR) alpha and beta chains.
Extracellular domain
As described herein, the CAR comprises a target-specific binding element, also referred to as an antigen binding domain or portion. The choice of domain depends on the type and number of ligands defining the target cell surface. For example, the antigen binding domain can be selected to recognize a ligand (e.g., a cancer antigen) that is a cell surface marker on a target cell that is associated with a particular disease state (e.g., cancer). Thus, examples of cell surface markers that can be ligands for the antigen binding domain in a CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.
In some embodiments, the extracellular domain of the CAR comprises an antigen binding agent that specifically binds to a cancer antigen. In certain embodiments, the CAR binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-associated embodiments described herein. Antigen sources include, but are not limited to, cancer proteins. The antigen may be expressed as a peptide or as an intact protein or as a portion thereof. The whole protein or a portion thereof may be native or mutagenized. Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA 1X), carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD 19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of Cytomegalovirus (CMV) infected cells (e.g., cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4 (erb-B2, 3, 4), folate Binding Protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD 2), ganglioside G3 (GD 3), guanylate cyclase (Guanyyl cyclase) C (GCC), HER 2 (ITER-2), human telomerase reverse transcriptase (hTERT), interleukin 13 receptor subunit alpha-2 (IL-L3 Rcx 2), k-light chain, kinase insert domain receptor (KDR), lewis Y (LeY), ll cell adhesion molecule (L1 CAM), melanoma antigen family A,1 (MAGE-A1), mucin 16 (MUC 16), mucin 1 (MUC 1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTl, GPl00, proteinase 3 (PR 1), tyrosinase, TERNT, ephA2, G2D ligand, cancer-testis antigen NY-ES0-1, tumor embryo antigen (h 5T 4), prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), PTK7 ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-l), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-l, CD70, tim3, CLEC12A and ERBB.
In certain embodiments, the CAR binds to a CD19 polypeptide. In certain embodiments, the CAR binds to a human CD19 polypeptide. In certain embodiments, the CAR binds to an extracellular domain of a CD19 protein. In certain embodiments, the CD19 CAR comprises the sequences provided in table 3.
In certain embodiments, the CAR binds to a GCC polypeptide. In certain embodiments, the CAR binds to a human GCC polypeptide. In certain embodiments, the CAR binds to an extracellular domain of a GCC protein. In certain embodiments, the anti-GCC CAR comprises the sequences provided in table 3.
In certain embodiments, the CAR binds to a mesothelin polypeptide. In certain embodiments, the CAR binds to a human mesothelin polypeptide. In certain embodiments, the CAR binds to an extracellular domain of mesothelin protein.
In certain embodiments, the CAR binds to a pathogen antigen, e.g., for use in treating and/or preventing a pathogen infection or other infectious disease in a subject, e.g., with reduced immune function. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites and protozoa capable of causing diseases.
Non-limiting examples of viruses include the retrovirus family (e.g., human immunodeficiency virus such as HIV-l (also known as HDTV-III, LAVE, or HTLV-IIELAV, or HIV-III), other isolates such as HIV-LP), the picornaviridae family (e.g., polioviruses, hepatitis A, enteroviruses, human coxsackieviruses, rhinoviruses, epstein-Barr virus (echoviruses)); the calixaviridae family (e.g., strains causing gastroenteritis), the togaviruses family (e.g., equine encephalitis, german measles virus), the flaviviridae family (e.g., dengue virus, encephalitis virus, yellow fever virus), the coronaviridae family (e.g., coronavirus), the baculoviruses family (e.g., vesicular stomatitis virus, rabies virus), the filoviruses family (e.g., ebola virus), the paramyxoviridae family (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus), the orthomyxoviridae family (e.g., viruses) the bunyaviridae (e) family (e.g., bunyaviridae, hantakiae, pseudoviridae), the flaviviridae (e) and the Paramygdalidae (e) virus (Parvoviridae), the flaviviridae (e), and the Paramyxoviridae (e) Polyoma virus); adenoviridae (most adenoviruses); herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus); poxviridae (smallpox virus, vaccinia virus, poxvirus); and iridoviridae (e.g., african swine fever virus); and unclassified viruses (e.g., pathogens of delta hepatitis (considered as a deletional satellite of hepatitis B virus), pathogens of non-a, non-B hepatitis (type 1 = internal transmission; type 2 = parenteral transmission (i.e., hepatitis c); norwalk and related viruses, and astroviruses).
Non-limiting examples of bacteria include Pasteurella, staphylococci, streptococci, E.coli, pseudomonas and Salmonella. Specific examples of infectious bacteria include, but are not limited to, helicobacter pylori, borrelia burgdorferi (Borelia burgdorferi), legionella pneumophila (Legionell a pneumophilia), mycobacteria (e.g., mycobacterium tuberculosis (M. Tuberculosis), mycobacterium avium (M. Avium), mycobacterium intracellulare (M. Introcelulae), mycobacterium kansasii (M. Kansaii), mycobacterium gordonae (M. Gordonae)), staphylococcus aureus, neisseria gonorrhoeae (Neisseria gonorrhoeae), neisseria meningitidis (Neisseria meningi tidis), listeria monocytogenes, streptococcus pyogenes (group A streptococcus), streptococcus agalactiae (group B streptococcus), streptococcus faecalis (grass green group), streptococcus faecalis, streptococcus bovis, streptococcus (anaerobic), streptococcus pneumoniae, campylobacter pathogenic bacteria, enterococcus, haemophilus influenzae, bacillus anthracis, corynebacterium, erysipelas, clostridium perfringens (Clostridium perfringers), candida, clostridium tetanus, enterobacter, klebsiella, streptococcus multiple pneumophila (3758), streptococcus (group A), streptococcus pyogenes (group A), streptococcus sp (group B streptococcus) and Leucomatous (group A), leptospira) and Leptospira (group A.parvum) of Leptospira (6543).
In certain embodiments, the pathogen antigen is a viral antigen present in Cytomegalovirus (CMV), a viral antigen present in epstein barr virus (Epstein Barr Virus, EBV), a viral antigen present in Human Immunodeficiency Virus (HIV), or a viral antigen present in influenza virus.
In certain embodiments, the extracellular domain of the CAR comprises a linker. In some embodiments, the linker comprises GGGGS (SEQ ID NO: 59). In some embodiments, a subject isThe joint comprises (GGGGS) n (SEQ ID NO: 59), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 61).
In some embodiments, the extracellular antigen-binding domain comprises an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab ' fragment, a F (ab ') 2 fragment, a Fd ' fragment, a Fd fragment, an isolated CDR, or a collection thereof; single chain variable fragments (scFv), polypeptide-Fc fusions, single domain antibodies (sdabs), camelized antibodies; masking antibodies, small modular immunopharmaceuticals ("SMIPsTM"), single chain, tandem diabodies, VHH, anti-cargo, nanobodies, humanized antibodies, minibodies, bites, ankyrin repeat proteins, DARPIN, avimer, DART, TCR-like antibodies, adnectin, affilin, penetrating antibodies; affinity antibody, trimerX, mini-protein, fynomer, centyrin; and KALBITOR, or a fragment thereof.
In some embodiments, the extracellular antigen-binding domain of the CAR comprises a single chain variable fragment (scFv). In some embodiments, the extracellular antigen-binding domain of the CAR comprises a single domain antibody (sdAb). In some embodiments, a single domain antibody (sdAb),
transmembrane domain
As described herein, the CAR comprises a transmembrane domain. With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular antigen binding domain of the CAR. The transmembrane domain may be derived from natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein.
The transmembrane region used in the CARs described herein can be derived from (i.e., comprise at least the following transmembrane regions) the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will predominantly comprise hydrophobic residues, such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In some embodiments, the linker is a glycine-serine duplex or an alanine triplet linker.
In some embodiments, a transmembrane domain that naturally associates with one of the domains of the CAR is used in addition to a transmembrane domain as previously described. In some embodiments, the transmembrane domains may be selected by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.
In some embodiments, the transmembrane domain in a CAR of the invention is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises nucleic acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO:42. In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO. 42. In some embodiments, the transmembrane domain comprises a sequence having at least one, two, or three modifications (e.g., substitutions) but NO more than 20, 10, or 5 modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO. 42, or a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 42.
In CARs, a spacer domain (also referred to as a hinge domain) can be disposed between an extracellular domain and a transmembrane domain, or between an intracellular domain and a transmembrane domain. Spacer domain means any oligopeptide or polypeptide used to connect a transmembrane domain with an extracellular domain and/or a transmembrane domain with an intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen binding fragment increases. Exemplary spacers are known to those of ordinary skill in the art and include those listed in U.S. patent nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and U.S. patent publication nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.
The spacer domain preferably has a sequence that promotes binding of the CAR to the antigen and enhances signaling into the cell. Examples of amino acids expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids may be used as amino acids constituting the spacer domain.
In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises nucleic acid sequence IEVMYPPPYLDNEKSNGTIIHV KGKHLCPSPLFPGPSKP (SEQ ID NO: 41). In some embodiments, the hinge domain comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO. 41. In some embodiments, the hinge domain comprises a sequence having at least one, two, or three modifications (e.g., substitutions) but NO more than 20, 10, or 5 modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO. 41, or a sequence at least 95-99% identical to the amino acid sequence of SEQ ID NO. 41.
In some embodiments, the hinge domain and the transmembrane domain are derived from the same molecule. In other embodiments, the hinge and transmembrane domains are derived from different molecules (e.g., CD8 fused to CD 28). In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises nucleic acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV GGVLACYSLLVTVAFIIFWV (SEQ ID NO: 43). In some embodiments, the hinge domain comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO. 43. In some embodiments, the hinge domain comprises a sequence having at least one, two, or three modifications (e.g., substitutions) but NO more than 20, 10, or 5 modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO. 43.
Intracellular domains
The cytoplasmic domain or intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of an immune cell in which the CAR has been placed. The term "effector function" refers to a specific function of a cell. For example, the effector function of T cells may be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to perform a particular function. Although it is generally possible to use whole intracellular signaling domains, in many cases it is not necessary to use whole chains. In the case of using a truncated portion of the intracellular signaling domain, such truncated portion can be used in place of the complete strand, so long as it is capable of transducing an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector functional signal.
Examples of intracellular signaling domains for CARs include cytoplasmic sequences of T Cell Receptors (TCRs) and co-receptors that work together to initiate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same function. The signal produced by TCR alone is not sufficient to fully activate T cells, a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via a TCR (primary cytoplasmic signaling sequence), and those that function in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequence).
The primary cytoplasmic signaling sequence modulates primary activation of the TCR complex either in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs disclosed herein that contain primary cytoplasmic signaling sequences particularly for CARs include those derived from TCR ζ (cd3ζ), fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, and CD66 d. Specific non-limiting examples of ITAMs include amino acid numbers 51 to 164 with CD3 ζ (NCBI RefSeq: np.sub.— 932170.1), amino acid numbers 45 to 86 with fcepsilon. RI. γ (NCBI RefSeq: np.sub.— 004097.1), amino acid numbers 201 to 244 with fcepsilon. RI. β (NCBI RefSeq: np.sub.— 000130.1), amino acid numbers 139 to 182 with CD3 γ (NCBI RefSeq: np.sub.— 000064.1), amino acid numbers 128 to 171 with CD3 δ (NCBI RefSeq: np.sub.— 000723.1), amino acid numbers 153 to 207 with CD3 epsilon (NCBI RefSeq: np.sub.— 000724.1), amino acid numbers 402 to 495 with CD5 (NCBI RefSeq: np.sub.— 055022.2), amino acid numbers 707 to 847 with NCBI RefSeq: np.sub.— 001762.2), amino acid numbers 166 to 166 with CD79a (NCBI RefSeq: np.sub.— 000064.1), amino acid numbers 166 to 39 with the same function as those of amino acid numbers of NCBI RefSeq (np.sub.— 000723.1), amino acid numbers 153 to 207 with CD3 δ (np.sub.— 001774.1, and variants thereof. Amino acid numbering based on the amino acid sequence signals of NCBI RefSeq ID or GenBank described herein is based on the full length of the precursor (including signal peptide sequences, etc.) of each protein. In one embodiment, the cytoplasmic signaling molecule of the CAR comprises a cytoplasmic signaling sequence derived from cd3ζ.
In some embodiments, the intracellular domain of the CAR may be designed to comprise the CD 3-zeta signaling domain itself, or to bind to any other desired cytoplasmic domain available in the context of the CAR. For example, the intracellular domain of the CAR may comprise a cd3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are necessary for lymphocytes to respond efficiently to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like. Specific non-limiting examples of such co-stimulatory molecules include peptides having the following sequences: amino acid numbers 236 to 351 of CD2 (NCBI Refseq: NP. Sub. - -001758.2), 421 to 458 of CD4 (NCBI Refseq: NP. Sub. - -000607.1), 402 to 495 of CD5 (NCBI Refseq: NP. Sub. - -055022.2), 207 to 235 of CD8 alpha (NCBI Refseq: NP. Sub. - -001759.3), 196 to 210 of CD83 (GenBank: AAA 35664.1), 181 to 220 of CD28 (NCBI Refseq: NP. Sub. - -006130.1), 214 to 255 of CD137 (4-1BB,NCBI RefSeq:NP.sub.- -001552.2), 241 to 277 of CD134 (OX 40, NCBI Refseq: NP. Sub. - -003318.1), and 166 to 199 of ICOS (NCBI Refseq: NP. Sub. - -036224.1), and variants thereof having the same function as these peptides. Thus, while the disclosure herein primarily uses 4-1BB as an example of a co-stimulatory signaling element, other co-stimulatory elements are within the scope of the disclosure.
The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a linkage. In some embodiments, the linker is a glycine-serine duplex or an alanine triplet linker.
In some embodiments, the intracellular domain is designed to comprise a CD28 costimulatory signaling domain. In some embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS (SEQ ID NO: 44).
In some embodiments, the intracellular domain is designed to comprise a signaling domain of CD3- ζ and a signaling domain of CD 28.
In some embodiments, the intracellular domain comprises CD3- ζ having one or more modified immune receptor tyrosine basal activation motifs (ITAMs). In some embodiments, the intracellular domain comprises CD3- ζ having three immunoreceptor tyrosine basal activation motifs (ITAMs), wherein the first is unmodified and the second and third ITAMs are altered, designated "1 XX". "in some embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to RVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIG MKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 45).
In some embodiments, the CAR comprises an intracellular signaling domain comprising a modified CD3z polypeptide (e.g., a modified human CD3z polypeptide) that comprises native ITAM1, native BRS2, native BRS3, ITAM2 variants with two loss-of-function mutations, and ITAM3 variants with two loss-of-function mutations, and a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide).
In another embodiment, the intracellular domain is designed to comprise a signaling domain of CD 3-zeta and a signaling domain of 4-1 BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domains of CD28 and 4-1 BB.
In some embodiments, the intracellular domain of the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3- ζ.
In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence provided in table 3.
TABLE 3 exemplary chimeric antigen receptors
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Functional features of CAR
The functional portions of the CARs disclosed herein are also expressly included within the scope of the present invention. The term "functional moiety" when used in reference to a CAR refers to any portion or fragment of one or more CARs disclosed herein that retains the biological activity of the CAR of the portion (parent CAR). Functional moieties encompass, for example, those portions of the CAR that remain similar to, to the same extent as, or to a greater extent than the parent CAR retains the ability to recognize a target cell or detect, treat, or prevent a disease. With respect to the parent CAR, the functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.
The functional moiety may comprise additional amino acids at the amino or carboxy terminus or both termini of the moiety that are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional moiety, e.g., identifying a target cell, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acid enhances biological activity as compared to the biological activity of the parent CAR.
Functional variants of the CARs disclosed herein are included within the scope of the disclosure. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR mutated thereby. Functional variants encompass those variants of a CAR (parent CAR) such as described herein that retain the ability to recognize target cells to a similar degree, to the same degree, or to a greater degree than the parent CAR. With respect to the parent CAR, the functional variant can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical to the amino acid sequence of the parent CAR.
The functional variant may, for example, comprise the amino acid sequence of the parent CAR, which has at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise an amino acid sequence of the parent CAR, which has at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions preferably do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent CAR.
The amino acid substitutions of the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid having a particular physical and/or chemical property is exchanged for another amino acid having the same or similar chemical or physical property. For example, the conservative amino acid substitution may be a substitution of an acidic/negatively charged polar amino acid with another acidic/negatively charged polar amino acid (e.g., asp or Glu), a substitution of an amino acid with a non-polar side chain with another amino acid with a non-polar side chain (e.g., ala, gly, val, he, leu, met, phe, pro, trp, cys, val, etc.), a substitution of a basic/positively charged polar amino acid with another basic/positively charged polar amino acid (e.g., lys, his, arg, etc.), a substitution of an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., asn, gin, ser, thr, tyr, etc.), a substitution of an amino acid with a beta-branched side chain with another amino acid with a beta-branched side chain (e.g., he, thr, and Val), a substitution of an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., his, phe, trp and Tyr), etc.
The CAR can consist essentially of one or more specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.
The CAR (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, provided that the CAR (or functional portion or functional variant thereof) retains its biological activity, e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, the CAR can be about 50 to about 5000 amino acids in length, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids in length.
The CAR (including functional parts and functional variants of the invention) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example: aminocyclohexane carboxylic acid, norleucine, -amino-N-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine β -hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N ' -benzyl-N ' -methyl-lysine, N ' -dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocycloheptane carboxylic acid, a- (2-amino-2-norbornane) -carboxylic acid, γ -diaminobutyric acid, β -diaminopropionic acid, homophenylalanine and a-tert-butylglycine.
The CAR (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and/or optionally dimerized or polymerized or conjugated.
Substitutions and variants
In some embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to increase the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to complete the final construct, provided that the final construct has the desired characteristics, such as antigen binding.
a) Substitution, insertion and deletion variants
In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs and FR. The amino acid side chain class is described further below. Amino acid substitutions may be introduced into the antibody of interest and the product subjected to a desired activity screen, such as antigen binding retention/improvement, reduced immunogenicity, or ADCC or CDC improvement.
Amino acids can be grouped according to common side chain characteristics:
(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;
(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;
(3) Acid: asp, glu;
(4) Alkaline: his, lys, arg;
(5) Residues that affect chain orientation: gly, pro;
(6) Aromatic: trp, tyr, phe.
Non-conservative substitutions require the exchange of members of one of these classes for another class.
One type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further investigation will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).
Alterations (e.g., substitutions) may be made in the HVR, for example, to increase antibody affinity. Such changes may be made in HVR "hot spots", i.e. residues encoded by codons that undergo high frequency mutations during somatic maturation (see e.g. chordhury, methods mol. Biol.207:179-196 (2008)), and/or SDR (a-CDRs), wherein the binding affinity of the resulting variant VH or VL is tested. Affinity maturation is achieved by construction and rescreening from secondary libraries, as described, for example, in Hoogenboom et al Methods in Molecular Biology 178:1-37 (O' Brien et al, human Press, totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced in the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In general, CDR-H3 and CDR-L3 are targeted in particular.
In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions provided herein) may be made within the HVR that do not substantially reduce binding affinity. Such changes may be outside of the HVR "hot spot" or CDR. In some embodiments of the variant VHH sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.
One useful method for identifying residues or regions in an antibody that can be targeted for mutagenesis is known as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this approach, a residue or group of residues of interest (e.g., charged residues such as Arg, asp, his, lys and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the original substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact site between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine if they contain the desired property.
Amino acid sequence insertions include amino and/or carboxy terminal fusions, ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., against ADEPT) or a polypeptide that increases the serum half-life of the antibody.
b) Glycosylation variants
In some embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. The addition or deletion of glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.
Where an antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched, double-antennary oligosaccharides, which are typically linked by an Asn 297N-bond to the CH2 domain of the Fc region. See, e.g., wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of a double-antennary oligosaccharide structure. In some embodiments, antibodies of the application may be oligosaccharide modified to create antibody variants with certain improved properties.
In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose (directly or indirectly) attached to an Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chains of Asn297, measured by MALDI-TOF mass spectrometry, as described for example in WO 2008/077546, relative to the sum of all sugar structures (e.g. complex, hybrid and highly mannose structures) attached to Asn 297. Asn297 refers to an asparagine residue at about position 297 of the Fc region (EU numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence differences in antibodies. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108 (Presta, l.); US2004/0093621 (Kyowa Hakko Kogyo co., ltd). Examples of disclosures relating to "defucosylation" or "fucose deficiency" antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US2002/0164328; US2004/0093621; US 2004/013321; US 2004/010704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al, arch. Biochem. Biophysi. 249:533-545 (1986), U.S. patent application No. US2003/0157108 A1, presta, L, and WO 2004/056312 A1, adams et al), and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004), kanda, y. Et al, biotechnol. Bioeng, 94 (4): 680-688 (2006), and WO 2003/085107).
Antibody variants further have bisected oligosaccharides, for example, wherein the double-antennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Maiset et al); U.S. Pat. No. 6,602,684 (Umana et al); US 2005/0123946 (Umana et al). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, s.); and WO 1999/22764 (Raju, S.).
The CAR (including functional parts and functional variants thereof) can be obtained by methods known in the art. The CAR may be prepared by any suitable method of preparing a polypeptide or protein. Suitable methods for de novo synthesis of polypeptides and proteins are described in the references, for example Chan et al, fmoc Solid Phase Peptide Synthesis, oxford University Press, oxford, united Kingdom,2000; peptide and Protein Drug Analysis, reid, r.m., marcel Dekker, inc.,2000; epitope Mapping, westwood et al, oxford University Press, oxford, united Kingdom,2001; and U.S. patent No. 5,449,752. In addition, polypeptides and proteins can be recombinantly produced using standard recombinant methods using the nucleic acids described herein. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual, 3 rd edition, cold Spring Harbor Press, cold Spring Harbor, N.Y.2001; and Ausubel et al Current Protocols in Molecular Biology, greene Publishing Associates and John Wiley & Sons, N Y,1994. In addition, some CARs (including functional portions and functional variants thereof) can be isolated and/or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans), and the like. Methods of isolation and purification are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be synthesized commercially by the factory. In this regard, the CAR can be synthetic, recombinant, isolated, and/or purified.
Detectable markers and tags
CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, antigen binding fragments thereof, can also be expressed (e.g., co-expressed) with a tag protein. In some embodiments, the furin recognition site and the downstream 2A ribosomal sequence are designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence. In some embodiments, the 2A sequence comprises the nucleic acid sequence GSGATNFSLLK QAGDVEENPGPSEQ ID NO:58. In some embodiments, the furin and P2A sequences comprise a nucleic acid sequence encoding the amino acid sequence SEQ ID NO. 58. In some embodiments, the P2A tag comprises the amino acid sequence of SEQ ID NO:58 or has a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.
CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, antigen binding fragments thereof, can also be conjugated to a detectable marker; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed Tomography (CT), computed Axial Tomography (CAT) scan, magnetic Resonance Imaging (MRI), magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic inspection, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, detectable markers that may be used include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine (rhodomine), 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green Fluorescent Protein (GFP), yellow Fluorescent Protein (YFP) may also be used. The CAR, CAR-expressing T cells, antibodies, or antigen binding portions thereof, can also be conjugated to enzymes for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the CAR, CAR-expressing T cells, antibodies, antigen binding fragments thereof, are conjugated to a detectable enzyme, detection can be performed by adding additional reagents that are used by the enzyme to produce a discernible reaction product. For example, when horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that is visually detectable. The CAR, CAR-expressing T cells, antibodies, or antigen binding portions thereof, can also be conjugated to biotin and detected via indirect measurement of avidin (avidin) or streptavidin (strepavidin) binding. It should be noted that the avidin itself may be conjugated to an enzyme or fluorescent label.
The CAR, CAR-expressing T cells, antibodies, or antigen binding fragments thereof, can be conjugated to a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide may also be used as labels. The antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. The antibody or antigen binding fragment may also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site of a secondary antibody, metal binding domain, epitope tag) that is recognized by a secondary reporter gene.
The CAR, CAR-expressing T cells, antibodies, or antigen binding fragments thereof may also be conjugated to radiolabeled amino acids. Radiolabels may be used for diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more antigens and cells expressing antigens disclosed herein by x-ray, emission spectroscopy, or other diagnostic techniques. In addition, the radiolabel may be therapeutically useful as a toxin to treat a tumor in a subject, such as to treat neuroblastoma. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I。
methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, a radiolabel may be detected using a photographic film or a scintillation counter, and a fluorescent marker may be detected using a photodetector to detect the emitted light. Enzymatic labels are typically detected by providing an enzyme with a substrate and detecting the reaction product of the enzyme acting on the substrate, and detecting the chromogenic label by simply observing the chromogenic label.
Immunoreactive cells and host cells
One aspect of the application provides an engineered immune effector cell (e.g., an immunoreactive cell). As used herein, "immunoreactive cells" refers to cells or progenitor cells or their progeny that play a role in an immune response. In some embodiments, the immunoreactive cells comprise an immunomodulatory system described herein (e.g., cells comprising a targeting agent specific for a tumor-associated antigen or stress ligand; and nucleic acid sequences encoding polypeptides that modulate TGF-beta signaling). In some embodiments, the immunoreactive cells comprise an immunoregulatory system described herein (e.g., a nucleic acid sequence encoding a chimeric antigen receptor (CAR; and a nucleic acid sequence encoding a polypeptide that modulates TGF- β signaling).
In some embodiments, the immune effector cells are T cells, NK cells, peripheral Blood Mononuclear Cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immunoreactive cell is a T cell.
For purposes herein, the T cell may be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., jurkat, supTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a variety of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. The T cells may be human T cells. The T cells may be T cells isolated from a human. The T cells may be any type of T cell and may be at any stage of development, including but not limited to: cd4+/cd8+ double positive T cells, cd4+ helper T cells (e.g., th1 and Th2 cells), cd8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., tscm), primitive T cells, and the like. The T cells may be cd8+ T cells or cd4+ T cells.
In one embodiment, a CAR as described herein can be used in a suitable non-T cell. Such cells are those having immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.
One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus or algae, or may be a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell, i.e. isolated directly from an organism such as a human. The host cell may be an adherent cell or a suspension cell (i.e., a suspension grown cell). Suitable host cells are known in the art and include, for example, DH 5. Alpha. E.coli cells, chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. When the host cell can be any cell type, can be derived from any type of tissue, and can be at any stage of development, the host cell can be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). The host cell may be a T cell.
One embodiment also provides a population of cells comprising at least one host cell described herein. The cell population may be a heterogeneous population comprising host cells comprising any of the recombinant expression vectors described, as well as at least one other cell (e.g., host cells (e.g., T cells) that do not comprise any of the recombinant expression vectors), or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population, wherein the population comprises predominantly (e.g., consists essentially of) host cells comprising the recombinant expression vector. The population may also be a clonal population of cells, wherein all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
CARs (including functional portions thereof and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen binding portions thereof) can be isolated and/or purified. For example, a purified (or isolated) host cell preparation is one in which the host cell is purer than the cell in its natural environment in vivo. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells comprise at least about 50%, such as at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, may be greater than about 60%, about 70% or about 80%, or may be about 100%.
Nucleic acid and expression vector
One embodiment of the invention further provides a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acids of the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains, and/or intracellular T cell signaling domains described herein.
In some embodiments, the nucleotide sequence may be codon modified. Without being bound by a particular theory, it is believed that codon optimization of the nucleotide sequence may increase the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve substitution of a native codon for another codon encoding the same amino acid, but translation may be achieved by a more readily available tRNA in the cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structure that may interfere with translation, thereby increasing translation efficiency.
In one embodiment of the invention, the nucleic acid can comprise a codon-modified nucleotide sequence encoding an antigen binding domain of a CAR of the invention. In another embodiment of the invention, the nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).
Expression vectors include plasmids, retroviruses, cosmids, YACs, EBV derived episomes, and the like. One convenient vector is a vector encoding a functionally complete human CH or CL immunoglobulin sequence, which has appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing typically occurs between the splice donor site inserted in the J region and the splice acceptor site prior to the human C region, and also occurs in the splice region within the human CH exon. Suitable expression vectors may comprise a number of components, such as an origin of replication, a selectable marker gene, one or more expression control elements such as transcriptional control elements (e.g., promoters, enhancers or terminators) and/or one or more translational signals, signal sequences, or leader sequences, and the like. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The resulting chimeric antibody may be linked to any strong promoter. Examples of suitable vectors that may be used include those suitable for mammalian hosts and based on viral replication systems, such as simian virus 40 (SV 40), rous Sarcoma Virus (RSV), adenovirus 2, bovine Papilloma Virus (BPV), papova BK mutant (BKV), or mouse and human Cytomegalovirus (CMV), as well as Moloney Murine Leukemia Virus (MMLV), the natural Ig promoter, and the like. A variety of suitable vectors are known in the art, including vectors maintained in single or multiple copies, or integrated into the host cell chromosome, e.g., via LTR, or via an artificial chromosome engineered to have multiple integration sites (Lindenbaum et al, nucleic Acids res.32:e172 (2004), kennard et al, biotechnol. Bioeng.2009, 5-month 20-day online). Additional examples of suitable carriers are listed in the following sections.
Accordingly, the invention provides one or more expression vectors comprising a nucleic acid encoding an antibody, an antigen-binding fragment of an antibody (e.g., a human, humanized, chimeric, or any of the foregoing), an antibody chain (e.g., heavy chain, light chain), or an antigen-binding portion of an antibody chain that binds tgfβ or tgfβr. In some embodiments, the invention provides one or more expression vectors comprising the extracellular domain of a nucleic acid of tgfβr.
Expression in eukaryotic host cells is useful because such cells are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies. However, any resulting antibody that is not active due to improper folding can be re-activated according to known methods (Kim and Baldwin, "Specific Intermediates in the Folding Reactions of Small Proteins and the Mechanism of Protein Folding", ann. Rev. Biochem.51, pages 459-89 (1982)). The host cell may produce a portion of an intact antibody, such as a light chain dimer or a heavy chain dimer, which is also an antibody analog according to the invention.
The invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a nucleic acid encoding a CAR construct described herein.
In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For the purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, allows the host cell to express the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide within the cell. The carrier does not naturally occur in one entity.
However, a portion of the vector may be naturally occurring. Recombinant expression vectors may comprise any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.
In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. The carrier may be selected from the group consisting of: pUC series (Fermentas Life Sciences, glen burn, md.), pBluescript series (Stratagene, laJolla, calif.), pET series (Novagen, madison, wis.), pGEX series (Pharmacia Biotech, uppsala, sweden) and pEX series (Clontech, palo Alto, calif.).
Phage vectors such as lambda, lambda ZapII (Stratagene), EMBL4 and lambda NMI 149 can also be used. Examples of plant expression vectors include pBIO1, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector or a lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including in particular self-inactivating lentiviral vectors as provided in Milone et al, mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, for example, but are not limited to, those from Oxford BioMedica plc Gene delivery technology, LENTIMAX from Lentigen TM Carrier systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
Many transfection techniques are generally known in the art (see, e.g., graham et al, virology,52:456-467 (1973); sambrook et al, supra; davis et al, basic Methods in Molecular Biology, elsevier (1986); and Chu et al, gene,13:97 (1981)).
Transfection methods include calcium phosphate co-precipitation (see, e.g., graham et al, supra), direct microinjection into cultured cells (see, e.g., capecchi, cell,22:479-488 (1980)), electroporation (see, e.g., shigekawa et al, bioTechniques,6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., mannino et al, bioTechniques,6:682-690 (1988)), lipid-mediated transduction (see, e.g., feigner et al, proc.Natl. Acad.Sci. USA,84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., klein et al, nature,327:70-73 (1987)).
In one embodiment, the recombinant expression vector may be prepared using, for example, sambrook et al, supra and Ausubel et al, supra, standard recombinant DNA techniques as described above. Constructs of circular or linear expression vectors can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, colE1, 2 μ plasmids, λ, SV40, bovine papilloma virus, and the like.
Recombinant expression vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, as appropriate, and considering whether the vector is DNA-based or RNA-based, it is specific for the type of host cell (e.g., bacterial, fungal, plant or animal) into which the vector is to be introduced. The recombinant expression vector may include restriction sites to facilitate cloning.
Recombinant expression vectors may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, etc. Marker genes suitable for use in the expression vectors of the present invention include, for example, a neomycin/G418 resistance gene, a hygromycin resistance gene, an histidinol resistance gene, a tetracycline resistance gene and an ampicillin resistance gene.
The recombinant expression vector may comprise a native or non-native promoter operably linked to, or complementary to or hybridizing to, a nucleotide sequence encoding the CAR (including functional portions and functional variants thereof). The choice of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the skilled artisan. Similarly, combinations of nucleotide sequences with promoters are also within the skill of the skilled artisan. The promoter may be a non-viral promoter or a viral promoter, such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or promoters found in the long terminal repeat of murine stem cell viruses.
Recombinant expression vectors can be designed for transient expression, stable expression, or both. In addition, recombinant expression vectors can be prepared for constitutive or inducible expression.
In addition, the recombinant expression vector may be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cell death that expresses the suicide gene. Suicide genes can be genes that confer sensitivity to an agent (e.g., a drug) on a cell expressing the gene and cause cell death when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, e.g., suicide Gene Therapy: methods and Reviews, springer, caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, sutton, surrey, UK), humana Press, 2004) and include, e.g., the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.
Therapeutic method
The present invention relates to methods of treatment comprising administering to a subject an anti-tgfβ, an anti-tgfβr antigen binding molecule, or an extracellular domain of tgfβr. In some embodiments, the CARs and antigen-binding molecules disclosed herein are useful in methods of treating or preventing a disease in a mammal. In this regard, one embodiment provides a method of treating or preventing cancer in a mammal comprising administering to the mammal an amount of a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and/or antigen-binding portion thereof, and/or pharmaceutical composition effective to treat or prevent cancer in the mammal.
In some embodiments, the CAR is expressed on donor cells, and these cells secrete an anti-tgfβ, an anti-tgfβr antigen binding molecule, or an extracellular domain of tgfβr. In some embodiments, the donor for T cell therapyT cells are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject that is not a patient (e.g., allogeneic T cell therapy). The car+ T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 4 Individual cells, at least about 10 5 Individual cells, at least about 10 6 Individual cells, at least about 10 7 Individual cells, at least about 10 8 Individual cells, at least about 10 9 Individual cells, or at least about 10 10 Individual cells.
In some embodiments, the therapeutically effective amount of T cells is about 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 Individual cells or about 10 8 Individual cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2X10 6 Individual cells/kg, about 3X10 6 Individual cells/kg, about 4X10 6 Individual cells/kg, about 5X10 6 Individual cells/kg, about 6X10 6 Individual cells/kg, about 7X10 6 Individual cells/kg, about 8X10 6 Individual cells/kg, about 9X10 6 Individual cells/kg, about 1X10 7 Individual cells/kg, about 2X10 7 Individual cells/kg, about 3X10 7 Individual cells/kg, about 4X10 7 Individual cells/kg, about 5X10 7 Individual cells/kg, about 6X10 7 Individual cells/kg, about 7X10 7 Individual cells/kg, about 8X10 7 Individual cells/kg, or about 9X10 7 Individual cells/kg. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is between about 1X10 6 Up to about 2X10 6 Between individual CAR positive live T cells/kg body weight up to a maximum dose of about 1x10 8 Individual CAR positive living T cells.
In some embodiments, the therapeutically effective amount of CAR-positive living T cells is about 0.25X10 6 To 2X10 6 Between them. In some embodiments, the therapeutically effective amount of CAR positive living T cells is 0.25x10 6 、0.3x10 6 、0.4x10 6 About 0.5x10 6 About 0.6x10 6 About 0.7x10 6 About 0.8x10 6 About 0.9x10 6 About 1.0x10 6 About 1.1x10 6 About 1.2x10 6 About 1.3x10 6 About 1.4x10 6 About 1.5x10 6 About 1.6x10 6 About 1.7x10 6 About 1.8x10 6 About 1.9x10 6 Or about 2.0x10 6 Individual CAR positive living T cells.
In some embodiments, the therapeutically effective amount of CAR-positive living T cells is about 0.4x10 8 Up to about 2x10 8 Between individual CAR positive live T cells. In some embodiments, the therapeutically effective amount of CAR positive living T cells is about 0.4x10 8 About 0.5x10 8 About 0.6x10 8 About 0.7x10 8 About 0.8x10 8 About 0.9x10 8 About 1.0x10 8 About 1.1x10 8 About 1.2x10 8 About 1.3x10 8 About 1.4x10 8 About 1.5x10 8 About 1.6x10 8 About 1.7x10 8 About 1.8x10 8 About 1.9x10 8 Or about 2.0x10 8 Individual CAR positive living T cells.
One embodiment further comprises subjecting the mammal to lymphatic depletion prior to administration of the CAR disclosed herein. Examples of lymphatic depletion include, but are not limited to: non-myeloablative lymphatic depleting chemotherapy, systemic irradiation, and the like.
For the purposes of the method in which the host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. In some embodiments, the cell is autologous to the mammal. In some embodiments, the cell is a mammalian allogeneic. As used herein, an allograft refers to any material derived from a different animal of the same species as the individual into which the material was introduced. When the genes at one or more loci are different, the two or more individuals are said to be allogeneic to each other. In some aspects, allogeneic material from individuals of the same species may be sufficiently genetically diverse to undergo antigenic interactions. As used herein, "autologous" refers to any material derived from the same individual into which it is later reintroduced.
The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to: rodentia mammals such as mice and hamsters, and lagomorpha mammals such as rabbits. The mammal may be from the order carnivora, including felines (cats) and canines (dogs). The mammal may be from the order artiodactyla, including cattle (cows) and pigs (pigs), or from the order artiodactyla, including horses (horses). The mammal may be of the order primates, simiales (Ceboids) or simiales (Simoids) (monkey) or of the order apes (human and ape). In some embodiments, the mammal is a human.
With respect to the method, the cancer may be any cancer, including acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder sarcoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal canal cancer or colorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulval cancer, chronic lymphocytic leukemia, chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (e.g., head and neck squamous cell carcinoma), hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-hodgkin's lymphoma, B chronic lymphocytic leukemia, hairy cell leukemia, acute Lymphoblastic Leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omentum cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.
In certain embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer has abnormal tgfβ expression or abnormal tgfβ signaling.
As used herein, the terms "treat" and "prevent" and derivatives thereof do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention, which one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the methods can provide for the treatment or prevention of any amount or any level of mammalian cancer.
In addition, the treatment or prevention provided by the methods may include treating or preventing a condition or symptom of one or more diseases (e.g., cancer) to be treated or prevented. Furthermore, for purposes herein, "preventing" may encompass delaying the onset of a disease or symptom or condition thereof.
Methods for testing CARs for their ability to recognize target cells and antigen specificity are known in the art. For example, clay et al, J.Immunol,163:507-513 (1999), teach methods for measuring cytokine (e.g., interferon-gamma, granulosa cell/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-alpha), or interleukin 2 (IL-2)) release. In addition, CAR function can be assessed by measuring cytotoxicity of cells as described in Zhao et al, J.Immunol,174:4415-4423 (2005).
Another embodiment provides the use of a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody or antigen-binding portion thereof and/or pharmaceutical composition of the invention for treating or preventing a proliferative disorder, such as cancer, in a mammal. The cancer may be any cancer described herein.
Any method of administration may be used for the disclosed therapeutic agents, including local and systemic administration. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The particular mode of administration and dosing regimen will be selected by the attending clinician, taking into account the details of the case (e.g., subject, disease state involved, and whether the treatment is prophylactic or not). Where more than one agent or composition is administered, one or more routes of administration may be used; for example, the chemotherapeutic agent may be administered orally, and the antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Methods of administration include injection, wherein the CAR, CAR T cell, conjugate, antibody, antigen binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier such as water, saline, ringer's solution, dextrose solution, 5% human serum albumin, non-volatile oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds may be used, for example, by applying an antibody or antigen binding fragment to a tissue region from which a tumor has been removed, or a region suspected of being predisposed to developing a tumor. In some embodiments, sustained intratumoral (or near tumor) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen binding fragment may be advantageous. In other examples, the conjugate is applied topically to the cornea as an eye drop, or intravitreally to the eye.
The disclosed therapeutic agents may be formulated in unit dosage forms suitable for precise dosage individual administration. Furthermore, the disclosed therapeutic agents may be administered in a single dose or multiple dose regimen. A multi-dose regimen is one in which the primary course of treatment may be more than one single dose, e.g., 1 to 10 doses, followed by additional doses at subsequent intervals as needed to maintain or potentiate the effect of the composition. Treatment may involve administration of the compound once a day or multiple times a day over a period of days to months or even years. Thus, the dosing regimen will also be determined based at least in part on the particular needs of the subject to be treated and will depend on the judgment of the administering physician.
Typical dosages of the antibody or conjugate may range from about 0.01 to about 30mg/kg, for example from about 0.1 to about 10mg/kg.
In particular examples, a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR T cell, or additional agent is administered to a subject in a multiple daily dosing regimen, e.g., for at least two consecutive days, 10 consecutive days, etc., e.g., for weeks, months, or years. In one example, the conjugate, antibody, composition, or additional agent is administered to the subject for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
In some embodiments, the disclosed methods comprise providing to the subject a surgical, radiation therapy, and/or chemotherapeutic agent in combination (e.g., sequentially, substantially simultaneously, or simultaneously) with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Methods and therapeutic dosages of such agents and treatments are known to those skilled in the art and can be determined by a skilled clinician. The preparation and dosing regimen of the additional agent may be determined empirically by the manufacturer's instructions or by the skilled practitioner. The preparation and dosing regimen of such chemotherapies is also described in Chemotherapy Service, (1992), m.c. perry, williams & Wilkins, baltimore, md.
In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Non-limiting examples of additional therapeutic agents that can be used with the combination therapy include microtubule binding agents, DNA intercalating or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene modulators, and angiogenesis inhibitors. These agents (which are administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with a CAR, CAR-T cell, antibody, antigen binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by a skilled clinician.
Other chemotherapeutic agents include, but are not limited to: alkylating agents such as nitrogen mustard (e.g., chlorambucil (chlorbamuicil), chlormethine (chlormethine), cyclophosphamide, ifosfamide, and melphalan (melphalan)), nitrosoureas (e.g., carmustine (carmustine), fotemustine (fotemustine), lomustine (lomustine), and streptozotocin (streptozocin)), platinum compounds (e.g., carboplatin, cisplatin (cislatin), oxaliplatin (oxaliptin), and BBR 3464), busulfan (busulfan), dacarbazine (dacarbazine), nitrogen mustard (mechlorethamine), procarbazine (procarbazine), temozolomide (temozolomide), thiotepa, and uramine (uramine); antimetabolites, such as folic acid (e.g., methotrexate (methotrexa), pemetrexed (pemetrexed), and raltitrexed (raltitrexed)), purines (e.g., cladribine (cloxaabine), clofarabine (fludarabine), mercaptopurine (mecaptopurine), and thioguanine (tioguaine)), pyrimidines (e.g., capecitabine (capecitabine)), cytarabine (cytarabine), fluorouracil (fluoroucil), and gemcitabine (gemcitabine); plant alkaloids, such as podophyllum (e.g., etoposide (etoposide) and teniposide (teniposide)), taxanes (e.g., docetaxel (docetaxel) and paclitaxel)), vinca (e.g., vinblastine (vinblastine), vincristine (vincristine), vindesine (vindeline) and vinorelbine (vinorelbine)); cytotoxic/antitumor antibiotics such as anthracycline family members (e.g., daunorubicin, doxorubicin (doxorubicin), epirubicin (epirubicin), idarubicin (epirubicin), mitoxantrone (mitoxantrone) and valrubicin), bleomycin (bleomycin), rifampicin (rifampicin), hydroxyurea and mitomycin; topoisomerase inhibitors such as topotecan (topotecan) and irinotecan (irinotecan); monoclonal antibodies, such as alemtuzumab (alemtuzumab), bevacizumab (bevacizumab), cetuximab (cetuximab), gemtuzumab (gemtuzumab), rituximab (rituximab), panitumumab (panitumumab), pertuzumab (pertuzumab), and trastuzumab (trastuzumab); photosensitizers such as aminolevulinic acid, methyl aminolevulinate, sodium poisson (porfimer sodium), and verteporfin (verteporfin); and other agents such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, dimidine interleukin (denileukin diftitox), erlotinib, estramustine, gefitinib, hydroxyformamide, imatinib, lapatinib, pazopanib, pranopalin, ma Suopu co-moroxydine, tolazamide, pranopalin, and sufentanib. The selection of such agents and the therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician.
The combination therapy may provide synergy and demonstrate synergy, i.e. an effect that results when the active ingredients are used together that is greater than the sum of the effects of the compounds when used separately. Synergistic effects can be achieved when the active ingredients are as follows: (1) Co-formulated and when administered or delivered simultaneously in a combined unit dose formulation; (2) when delivered alternately or in parallel as separate formulations; or (3) by some other scheme. When delivered in an alternating fashion, a synergistic effect may be achieved when the compounds are administered sequentially or delivered sequentially, for example by different injections in different syringes. Generally, during alternation, the effective dose of each active ingredient is administered sequentially, while in combination therapy, the effective doses of two or more active ingredients are administered together.
In various embodiments, the immune modulation system comprising a modulator of tgfβ signaling as described herein may be included in a treatment regimen that further includes administration of at least one additional agent to a subject. In various embodiments, the additional agent administered in combination with an immune modulation system comprising a modulator of tgfβ signaling as described herein may be a chemotherapeutic agent. In various embodiments, the additional agent administered in combination with an antigen binding agent as described herein may be an agent that inhibits inflammation.
In some embodiments, the tgfβ signaling modulator is a single domain antibody or a secreted scFv specific for human tgfβ. In some embodiments, the tgfβ signaling modulator is a single domain antibody or a secreted scFv specific for human tgfβr. In some embodiments, the tgfβ signaling modulator may be conjugated (e.g., linked) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) to bind to, deliver the therapeutic agent to, and kill cancer cells that express human tgfβ. In some embodiments, a modulator of tgfβ signaling is linked to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, cytokine, radioactive atom, siRNA or toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the agent is a radioactive atom.
In some embodiments, the methods may be performed in combination with other therapies for a tgfβ signaling abnormality disorder. For example, the composition may be administered to the subject prior to or concurrently with chemotherapy. In some embodiments, the composition may be administered to the subject concurrently with, prior to, or subsequent to the adoptive cell therapy.
In various embodiments, additional agents administered in combination with the immune modulation system comprising a modulator of tgfβ signaling as described herein may be administered at the same time, on the same day, or on the same week as the tgfβ signaling modulator. In various embodiments, additional agents administered in combination with a tgfβ signaling modulator described herein may be administered with the immune modulating system in a single formulation. In certain embodiments, the additional agent is administered in a manner that is temporally separate from the administration of the tgfβ signaling modulator as described herein, e.g., one or more hours, one or more days, one or more weeks, one or more months before or after, one or more weeks before or after the administration of the tgfβ signaling modulator. In various embodiments, the frequency of administration of one or more additional agents may be the same, similar, or different than the frequency of administration of a tgfβ signaling modulator as described herein.
In some embodiments, the compositions may be formulated with one or more additional therapeutic agents, e.g., additional therapies to treat or prevent a tgfβ -related disorder (e.g., cancer or autoimmune disorder) in a subject. Additional agents for treating a tgfβ -related disorder in a subject will vary depending on the particular disorder to be treated, but may include, but are not limited to, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, ifosfamide, carboplatin, etoposide, dexamethasone (dexamethasone), cytarabine, cisplatin, cyclophosphamide, or fludarabine.
Composition and method for producing the same
Provided herein are compositions for gene therapy, immunotherapy, and/or cell therapy comprising one or more of the disclosed CARs or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells (which specifically bind to one or more antigens disclosed herein) in a carrier (e.g., a pharmaceutically acceptable carrier). The composition may be prepared in unit dosage form for administration to a subject. The amount and time of administration is at the discretion of the attending clinician to achieve the intended result. The composition may be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CAR or CAR-expressing T cells, antibodies, antigen binding fragments, conjugates are formulated for parenteral administration, e.g., intravenous administration. Compositions comprising a CAR or CAR-expressing T cell, conjugate, antibody or antigen binding fragment as disclosed herein can be used, for example, in the treatment and detection of tumors, such as, and not limited to, neuroblastomas. In some examples, the compositions are useful for treating or detecting cancer. Compositions comprising a CAR or a CAR-expressing T cell, conjugate, antibody or antigen binding fragment as disclosed herein are also useful, for example, in detecting pathological angiogenesis.
The composition for administration can include a solution of the CAR or CAR-expressing T cells, conjugates, antibodies, or antigen binding fragments in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable materials. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvants, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of CAR or CAR-expressing T cells, antibodies or antigen binding fragments or conjugates in these formulations can vary widely and will be selected primarily according to the particular mode of administration selected and the needs of the subject, primarily based on fluid volume, viscosity, body weight, and the like. The actual methods of preparing such dosage forms for gene therapy, immunotherapy and/or cell therapy are known or will be apparent to those skilled in the art.
Typical compositions for intravenous administration include from about 0.01 to about 30mg/kg of antibody or antigen-binding fragment or conjugate (or CAR, or T cells expressing CAR, or corresponding dose of conjugate including antibody or antigen-binding fragment) per subject per day. Practical methods for preparing the administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, the Remington's Pharmaceutical Science, 19 th edition, mack Publishing Company, easton, pa. (1995) publication.
The controlled release parenteral formulations may be formulated as implants, oily injection solutions or as microparticle systems. For a broad overview of protein delivery systems, see Banga, A.J., therapeutic Peptides and Proteins: formulation, processing, and Delivery Systems, technomic Publishing Company, inc., lancaster, pa., (1995). Microparticle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or a drug, as the central core. In the microspheres, the therapeutic agent is dispersed throughout the particles. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. The capillaries have a diameter of about 5 μm, so only nanoparticles can be administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., kreuter, j., colloidal Drug Delivery Systems, j.kreuter, marcel Dekker, inc., new York, n.y., pages 219-342 (1994); and Tice and Tabi, treatise on Controlled Drug Delivery, A.Kydonieus, marcel Dekker, inc. New York, N.Y., pages 315-339 (1992).
The polymer can be used for ion-controlled release of the CAR or CAR-expressing T cells, antibodies, or antigen binding fragments, or conjugate compositions disclosed herein. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, account chem. Res.26:537-542, 1993). For example, the block copolymer polaxamer407 exists as a viscous but fluid liquid at low temperature, but forms a semi-solid gel at body temperature. It has been demonstrated as an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al, pharm. Res.9:425-434,1992; and Pec et al, J. Parent. Sci. Tech.44 (2): 58-65,1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of protein (Ijntema et al, int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al Liposome Drug Delivery Systems, technomic Publishing Co., inc., lancaster, pa. (1993)). Various additional systems for controlled delivery of therapeutic proteins are known (see U.S. patent nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).
Medicine box
In one aspect, kits using the CARs disclosed herein are also provided. For example, a kit for treating a tumor in a subject, or making CAR T cells expressing one or more CARs disclosed herein. The kit will typically include an antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell as disclosed herein. More than one disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell may be included in the kit.
The kit may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of various materials such as glass or plastic. The container typically houses a composition comprising one or more of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In several embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag, or a vial with a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat a particular disorder.
The label or package insert will typically further include instructions for use of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells, e.g., in a method for treating or preventing a tumor or manufacturing CAR T cells. The package insert typically includes instructions that are routinely contained in commercial packages of therapeutic products that contain information regarding the indication, usage, dosage, administration, contraindications and/or warnings of using such therapeutic products. The instructional material may be written, in electronic form (e.g., a computer hard disk or optical disk) or in visual form (e.g., a visual file). The kit may also include additional components to facilitate a particular application of the kit design. Thus, for example, the kit may additionally contain means for detecting a label (e.g., an enzyme substrate for enzyme labeling, a filter set for detecting fluorescent labels, a suitable secondary label (e.g., a secondary antibody), etc.). The kit may additionally include buffers and other reagents conventionally used to carry out particular methods. Such kits and suitable contents are well known to those skilled in the art.
Unless otherwise defined, all technical and scientific terms and phrases used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.
Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reaction and purification techniques may be carried out according to the manufacturer's instructions or as is commonly done in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989)), which is incorporated herein by reference for any purpose.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The invention will be more fully understood by reference to the following examples.
Examples
These examples are presented to aid in the understanding of the invention but are not intended to, and should not be construed to, limit its scope in any way. The examples do not include detailed descriptions of conventional methods (molecular cloning techniques, etc.) known to those of ordinary skill in the art.
Example 1 immunoreactive cells co-expressing Chimeric Antigen Receptor (CAR) and a modulator of TGF-B signaling
This example illustrates the use of the immune modulating system to co-express a TGF- β signaling modulator and a CAR in human T cells. Immunomodulatory constructs encoding TGF-B signaling modulators (e.g., anti-tgfβ and anti-tgfβr2) and anti-human CD19 CAR (SJ 25C1 extracellular antigen binding domain) are packaged for retroviral delivery. The Phoenix A retrovirus packaging cell line (ATCC) was grown to 50-70% confluence in DMEM 20% FBS and penicillin/streptomycin. DNA complexes were prepared using the respective plasmids encoding TGF-B signaling modulators and CAR constructs, helper plasmid gag-pol, pvvg and transduction reagent Fugene HD (Promega) according to the manufacturer's protocol. After 20-48 hours of transfection, virus supernatant was collected, aliquoted and frozen for further use.
Human PBMCs were isolated from Leukopaks using a density gradient and frozen until further use. Human T cells were isolated by magnetic selection from previously frozen PBMC (T cell isolation kit; stemcell). Purified human T cells were cultured for 2 days in complete Optimizer medium (Optimizer basal medium (ThermoFisher#A10221-01) +26ml OptiMizer supplement (ThermoFisher#A10484-02) +20ml ICSR (CTS immune cell SR), thermoFisher#A 25961-01) +10ml 200mM L-glutamine, (Gibco 25030-081) +PenStrep, (Gibco 15140-122)) containing 2ng/ml human IL-2 (Miltenyi) and T cell Transact beads (Miltenyi).
T cells were transferred to a plate coated with retronectin (Takara; 40ug/ml retronectin) and transduced with appropriate viral load. Transduction was confirmed and quantified by flow cytometry at different time points. Briefly, cells were incubated with 250ng of hCD19-hFc protein (RnD Systems) or internally produced hGC-Fc protein in FACS buffer for 1 hour at 4' C. After washing with FACS buffer, cells were resuspended in secondary antibodies against human FC (Biolegend) for 20 min at room temperature. Antibodies against CD4, CD8 or other surface markers were added in some experiments. Dead cells were excluded from analysis using a fixable vital dye (thermofilter). Cells were fixed in PBS2% FCS 4% formaldehyde prior to analysis by flow cytometry (FACS Fortessa, BD Biosciences). Transduction efficiency was shown as% viable cells positive for CAR staining. Flow cytometry results showed that populations of 87.6% lymphocytes, 76.1% single cells, 78.3% live cd3+ cells, and 75.8% cells showed CAR expression (fig. 1A-1D). Transduction efficiency was shown as viable cells positive for CAR staining (fig. 1E).
Example 2 in vitro killing Using TGF-beta regulatory human CAR-T cells
This example illustrates in vitro killing by human CAR-T cells that co-express a modulator of TGF- β signaling. In vitro killing of armored human CAR-T cells is comparable to unarmored CAR-T cells.
Raji (ATCC CD19 positive) or Raji CD19ko (human CD19 negative) were stained with the proliferation dye efluor 450 (thermofiser) and plated in 96-well plates for at least 2 hours according to the manufacturer's protocol, after which TGF- β regulatory CAR-T cells as described in example 1 were added. CAR-T cells were added at a ratio of effector to target of 0:1, 0.3:1, 1:1, 3:1, 9:1 to T cell addition alone and incubated overnight at 37 ℃. The following day, FACS staining was performed using fluorescent dye conjugated antibodies against human CD107a (LAMP-1) (bioleged), TCR alpha/beta (bioleged) and human CD4 antibodies (bioleged). Cells were incubated with antibody for 30 minutes at 4℃according to the manufacturer's protocol, washed with PBS, and stained with a fixable vital dye (thermofiser). Cells were washed with 1x Annexin V binding buffer (Biolegend) and stained with Annexin V FITC. Cells were fixed in cytofix (BD Biosciences) and then harvested on FACS Fortessa (BD Biosciences). TGF-beta regulated CD19 CAR-T cells exhibited target-specific in vitro killing on CD19 positive Raji cells (FIG. 2A), but not on CD19 negative control cells (Raji CD19 ko) (FIG. 2B).
EXAMPLE 3 secretion of TGF-beta modulators by immunoreactive cells
This example demonstrates that TGF-beta regulatory CAR-T cells secrete co-expressed TGF-beta modulators (e.g., anti-TGF-beta bound to TGF-beta and anti-TGF beta R2 bound to TGF beta R2).
Supernatants from TGF-beta regulatory CAR-T cells were assayed by ELISA to detect anti-TGF-beta and anti-TGF-beta R2 antibodies. Maxisorp 96-well plates were coated overnight at 4℃with 100. Mu.l of recombinant human TGF-. Beta.s (4; rnD System. Mu.g/ml) or hTGF-. Beta.R2-Fc (0.1 mg/ml; rnD System) in coating buffer. Plates were washed with 1x wash buffer and blocked with reagent diluent for 1 hour at room temperature. The CAR-T supernatant, recombinant tgfβr2-flag, or recombinant tgfβ -flag antibody was added and incubated for 2 hours at room temperature.
After another washing step, HRP-conjugated flag-tagged antibody was added and incubated for 30 minutes at room temperature. Plates were washed and TMB substrate was added for 10-20 minutes. The reaction was stopped using a stop reagent and the plates were read at 450nm using a Pherastar plate reader. ELISA using coated TGF-b detected high levels of TGF-b scFv VH-VL1 and TGF-b scFv VH-VL2 (FIG. 3A) compared to detection of binders with anti-flag tagged HRP antibodies with TGF-b scFv VL-VH. ELISA using coated TGFbR2-Fc detected high levels of TGFbR2 scFv VH-VL, TGFbR2 scFv VL-VH and hTGFbR2 VH1 from human CAR-T, but no mTGFbR2 VH1 (FIG. 3B). TGF-beta binders and TGF-beta R2 binders are secreted by TGF-beta regulatory CAR-T cells and bind to their cognate antigens.
Example 4 human CAR-T cells secrete neutralizing antibodies against TGF-beta/TGF-beta R2
This example illustrates the presence of neutralizing antibodies to TGF-beta/TGF-beta R2 in the TGF-beta regulated CAR-T supernatant.
Functional assessment of TGF- β blocking binders in CAR-T cell supernatants was performed using SBE-Luc reporter cells (HEK 293 cells expressing firefly luciferase under control of Smad Binding Elements (SBE) (BPS Biosciences), which were designed to monitor activity of TGF- β/Smad signaling pathways. TGF-beta proteins bind to their cognate receptors on the cell surface, triggering a signaling cascade that results in phosphorylation and activation of SMAD2 and SMAD3, and then forming a complex with SMAD 4. The SMAD complex translocates to the nucleus and binds to SMAD Binding Elements (SBEs), resulting in transcription and expression of TGF- β/SMAD responsive genes. The presence of blocking binders was detected by their ability to inhibit TGF-beta inducible luciferase expression in SBE-Luc reporter cells. An exemplary assay for evaluating the efficacy of inhibiting TGF-beta inducible reporter activity is performed as follows.
1X10 in 100. Mu.l fresh medium (X-VIVO 15 with 1X penicillin/streptomycin) per well 5 Concentration of individual cells SBE-Luc cells were seeded into poly-D lysine coated 96-well plates and incubated at 37℃and 5% CO 2 Incubate for 4 hours. The supernatant from the CAR-T cells or a dilution thereof was mixed with an equal volume of TGF- β (4 ng/ml in X-VIVO 15) and incubated for 15 minutes at room temperature to complex TGF- β with TGF-b contained in the CAR-T supernatant. Mu.l of the mixture was added to SBE-Luc reporter cells in duplicate and incubated at 37℃with 5% CO 2 Incubate overnight. The final concentration of TGF-beta was 1ng/ml. In the presence of 1ng/ml TGF-beta, each experiment included titration curves for incremental dilutions of TGF-beta antibody (1D 11 (BioXcell) or TGF-beta binder or TGF-beta R2 binder).
The next day, 100. Mu.l of culture supernatant was removed and 100. Mu.l of Luciferin-D containing medium was addedDetection reagent (ONE-Step) TM Luciferase assay system). Cells were resuspended and transferred to a white assay plate and luminescence measured using a Pherastar plate reader. Luciferase activity was recorded as CPM. Data were analyzed using MS Excel or Graphpad prism. Nonlinear regression fits were performed using the sigmoidal dose-response (variable slope) of Graphpad prism. IC50 values were calculated.
The inhibition activity (%) was calculated using the following equation:
inhibition (%) = (CPM of 1-sample/maximum CPM of TGF- β (1 ng/ml) -treated sample) X100
The results show that the supernatant from the CAR-T cells secreting constructs TGF-beta scFv VH-VL1 (SEQ ID NO: 1) and TGF-beta scFv VH-VL2 (SEQ ID NO: 2) inhibited TGF-beta signaling (FIG. 4). Additional constructs were designed and luciferase reporter assays were used to screen for secretion of multimeric binders against TGF- β or tgfβr2 (fig. 5 and 6). TGF-beta regulatory CAR-T cells that secrete multimeric antibodies against TGF-beta and TGF-beta R2 are identified. Regardless of the linker, human CAR-T cells can secrete multimeric TGF-b binders and inhibit TGF-b signaling. Four different linkers were analyzed as shown in the following figures. Similar results were observed with anti-GCC CAR-T cells (data not shown).
EXAMPLE 5 multimeric binding partners against TGF-beta or TGF-beta R2 secreted by TGF-B regulatory CAR-T cells
This example illustrates the screening and identification of mouse CAR-T cells that secrete multimeric binders against TGF- β or tgfβr2. To generate mouse CAR-T cells, the Platinum-E retrovirus packaging cell line was grown to 50-70% confluence in DMEM 20% FBS and penicillin/streptomycin. DNA complexes were prepared using immune modulating system plasmids encoding CAR constructs and TGFB modulators (e.g., anti-TGF-b scFv monomers, anti-TGF-b scFv dimers), packaging constructs, and transduction reagent Fugene HD according to the manufacturer's protocol. The solutions were mixed and incubated at room temperature for 10 minutes, and every 10cm 2 The cell dish was added with 850. Mu.l of complex. Mouse T cells were isolated from Balb/C or C57BL/6 mouse spleens using a T cell isolation kit for magnetic selection, respectively. Activation of purified mouse T cells with mouse T cellsBeads (1:1 ratio) were incubated in RPMI 10% heat-inactivated FCS, penicillin/streptomycin and mouse IL-2 (30U/ml) for 2 days. Viruses were collected about 48 hours after transfection and filtered through a 0.4 μm syringe filter. T cells were transferred to plates coated with retronectin (40 μg/ml retronectin coated according to manufacturer's protocol) and transduced with appropriate viral load. Transduction was confirmed and quantified by flow cytometry at different time points.
Cells were incubated with 250ng of hCD19-hFc protein in FACS buffer for 1 hour at 4 ℃. After washing with FACS buffer, cells were resuspended for 20 min at room temperature with secondary antibodies to human FC. In some experiments, antibodies against CD4, CD8 or other surface markers were added. Dead cells were excluded from analysis using fixable vital dyes. Cells were fixed in PBS2% FCS, 4% formaldehyde prior to analysis by flow cytometry (FACS Fortessa). Transduction efficiency is shown as the percentage (%) of viable cells positive for CAR staining.
Flow cytometry results showed relative proportions of non-armored T cells, TGF- β monomers, TGF- β dimers, and non-transduced cells. Supernatants from mouse CAR-T cells collected after transduction were probed for TGF-b signaling inhibition in SBE-Luc TGF-b reporting assays (method described in example 4). Based on luciferase reporter activity, supernatant from mouse CAR-T secreting TGF-b scFv monomers and dimers inhibited TGF-b signaling. Mouse CAR-T cells that secrete multimeric antibodies against TGF- β and tgfβr2 were identified.
Supernatants were collected two days post transduction and frozen at-80' C until used in ELISA. ELISA was performed as described in example 3. Human recombinant TGFbR2-Fc protein was used to capture the anti-human TGFbR2 binding-molecule, and mouse recombinant TGFbR2-Fc protein was used to detect the binding of TGFbR2 binding-molecule to mouse TGFbR 2. Binding was detected using anti-flag HRP antibodies with individual substrates. As shown in fig. 7, the binders hTGFbR2-VH2 and hTGFbR2-VH3 monomer and dimer secretion and TGFbR2 scFv VH-VL monomer and dimer binding to human TGFbR 2. No binding partner in the supernatant tested would bind to mouse TGFbR2 confirming the specificity for human TGFbR 2.
Example 6 in vivo anti-tumor efficacy of TGF-beta Signal transduction modulators secreting CAR-T cells
This example demonstrates the in vivo anti-tumor efficacy of CAR-T cells secreting anti-TGF- β mabs. Mouse armored CAR-T cells (co-expressing anti-human CD19 CAR with TGFb signaling modulator) inhibited syngeneic EMT6-hCD19 tumor growth better than non-armored CAR-T cells. In addition, the armored CAR-T cells reduce liver and lung metastasis. An EMT6 breast cancer cell line that overexpresses human CD19 and firefly luciferase as CAR-T target antigens was generated. EMT6 cells were transduced with a puromycin resistant virus harboring a plasmid encoding human CD19 under the control of the EF1a promoter. Puromycin was used to positively select EMT6-hCD19 cells and further purified by FACS sorting. EMT6-hCD19 cells were treated with a neomycin-resistant (Amsbio) virus at 5X10 in the presence of polybrene with a plasmid encoding firefly luciferase under the control of the EF1a promoter 7 IFU/mL transduction; moi=10. EMT6-hCD19-Fluc cells were positively selected using G418 (500. Mu.g/ml).
At 0.2x10 6 Individual live EMT6-hCD19-Fluc tumor cells were inoculated into mammary fat pads (in situ) of 6-16 week old female Balb/c mice (Jackson Labs). After 6 days of implantation, the tumor size reached about 50mm 3 And mice were randomly grouped to have similar average tumor sizes (average about 50mm 3 ) Is combined with cyclophosphamide (CPA; 200mg/kg i.p.) treatment. The next day, 500,000 mouse CAR-T cells from the isotype CD45.1 Balb/c mice were injected into the tail vein. Group 1 received non-transduced T cells, group 2 received CAR-T cells, and group 3 received CAR-T cells secreting anti-TGF- β scFv VH-VL 1. Body weight was measured twice weekly to monitor toxicity.
Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) =length x width x height x0.5236 (fig. 8). Any tumor was beyond 2000mm 3 Or mice with ulcerative tumors. Assessment of reduction in tumor size compared to control mice injected with non-transduced T cellsAntitumor effect. A complete responder is defined as a mouse without any detectable tumor.
CAR-T cells secreting TGF- β binders show high anti-tumor efficacy relative to non-armored CAR-T or non-transduced CAR-T cells. Liver and lung tumor cells expressing firefly luciferase were imaged by injection of luciferin and imaging by IVIS. D-luciferin solution (D-luciferin, vivo Glo TM Potassium fluorescein salt) was prepared at 15mg/ml and used at 150 mg/kg. Mice treated with CAR-T cells secreting TGF- β binders were able to reduce liver and lung metastasis (fig. 8A-8E).
Mouse CAR-T against human CD19, which secreted inhibitory antibodies against TGF- β (TGF-b scFv VH-VL 1), inhibited syngeneic EMT6-hCD19 tumor growth better than non-armored CAR-T cells and reduced liver and lung metastasis. The number of CAR-T cells has been previously titrated to obtain a suboptimal effect on non-armored CAR-T to identify improved activity of armored CAR-T cells.
Example 7 armored mouse CAR-T cells secreting TGFbR2 extracellular Domain (ECD) inhibit TGFb signaling Guide rail
SBE-Luc TGF- β reporting assays were performed that compared supernatants from armored mouse CAR-T secreting different TGF- β ligand traps (TGF- β scFv VH-VL1 to TGFbR2 ECD monomers, homodimers (fig. 9A) and heterodimers (fig. 9B)) with supernatants from unarmored CAR-T. SBE-Luc TGF-beta report assay showed very good inhibition of supernatant from armored mouse CAR-T against human CD19 secreting TGF beta R2 extracellular domain (ECD) dimer (but no secretion of monomer) and also showed comparable inhibition of secretion of TGF-beta scFv VH-VL1 dimer. Supernatants were collected 2 days post transduction. Tgfβr2ecd heterodimer inhibition including tgfβr2ecd and tgfβr1ecd was evaluated. TGF-beta R2 ECD heterodimers that inhibit TGF-b signaling were identified to be more potent than TGF-beta scFV VH-VL 1. Exemplary tgfβr2ecd sequences are shown in table 4.
TABLE 4 exemplary TGF beta R2 ECD sequences
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Example 8 anti-tumor efficacy of armored CAR-T cells secreting modulators of TGF-beta signaling
This example demonstrates the relative anti-tumor efficacy of CAR-T cells secreting anti-tgfβ mAb or tgfβr2-ECD in vivo.
Mouse CAR-T secreting tgfβr2ecd1+2 dimer showed improved anti-tumor effects in vivo compared to non-armored CAR-T cells. At 0.2x10 6 Individual live EMT6-hCD19-Fluc tumor cells were inoculated into mammary fat pads (in situ) of 6-16 week old female Balb/c mice (Jackson Labs). After 6 days of implantation, tumor size reached about 50mm3 and mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm3; n=8 per group) and treated with cyclophosphamide (CPA; 200mg/kg i.p.). The next day, 2 million mouse CAR-T cells from the isotype CD45.1 Balb/c mice were injected into the tail vein. Group 1 received non-transduced control T cells, group 2 received non-armored CAR-T cells, group 3 received CAR-T cells secreting tgfbr1+2ECD dimer, and group 4 received systemic anti-TGF- β antibody (clone 1d11.16.8;10mg/kg; 3 injections per week; i.v.). Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) Length x width x height x0.5236. Following the animal health protocol of the association, any tumor is brought to over 2000mm 3 Or mice with ulcerative tumors. Antitumor efficacy was assessed as a decrease in tumor size compared to control mice injected with non-transduced T cells. A complete responder is defined as a mouse without any detectable tumor.
Mouse CAR-T against human CD19 that secreted TGF- β ligand trap (mTGFbR 2 ecd1+2 dimer 1) inhibited syngeneic EMT6-hCD19 tumor growth better than non-armored CAR-T cells, which induced 3 complete responses compared to incomplete responses in control mice that received non-armored CAR-T or non-transduced T cells, or were treated with systemic anti-TGF- β antibodies (1D 11, 10mg/kg, 3x i.v. weekly). (FIG. 10).
Example 9 armored CAR-T cells secreting modulators of TGF-beta signaling in syngeneic tumor models Antitumor efficacy in (MC 38-hCD 19)
This example demonstrates the relative anti-tumor efficacy of CAR-T cells secreting anti-tgfβ mAb or tgfβr2-ECD in vivo. Mouse CAR-T cells armored with anti-TGF-b mAb (TGF-b scFv VH-VL 1) have improved effects in different syngeneic tumor models (MC 38-hCD 19).
MC38 colorectal cancer cell lines overexpressing human CD19 and firefly luciferase as CAR-T target antigens were generated and used for imaging. Briefly, MC38 cells were transduced with a puromycin resistant (CD19_FL_WT_pLVX-EF 1 a-IRES-Puro) virus harboring a plasmid encoding human CD19 under the control of the EF1a promoter. MC38-hCD19 cells were positively selected using puromycin. MC38-hCD19 cells were transduced with a virus harboring a plasmid encoding firefly luciferase under the control of the EF1a promoter and neomycin resistance (Amsbio, catalog number LVP435-PBS, 5x 10A 7IFU/mL, MOI=10) in the presence of polybrene. MC38-hCD19-Fluc cells were positively selected using G418 (geneticin).
At 0.2x10 6 The individual live MC38-hCD19-Fluc tumor cells were inoculated subcutaneously into 6-16 week old female C57BL/6 mice (Jackson Labs). Seven days after implantation, the tumor size reached about 50mm3 and the mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm3; n=8 per group) and treated with cyclophosphamide (CPA; 200mg/kg i.p.). The next day, 100,000 mouse CAR-T cells (or non-transduced T cells as negative controls) from the isotype CD 45.1C 57BL/6 mice were injected into the tail vein. Group 1 received non-transduced T cells. Group 2 received non-armored CAR-T cells. Group 3 received anti-TGF-beta secreting CAR-T cells (TGF-b scFv VH-VL 1). Weekly measurements Body weight was twice monitored for toxicity. Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) Length x width x height x0.5236. Following the animal health protocol of the association, any tumor is brought to over 2000mm 3 Or mice with ulcerative tumors. Antitumor efficacy was assessed as a decrease in tumor size compared to control mice injected with non-transduced T cells. A complete responder is defined as a mouse without any detectable tumor.
CAR-T cells secreting inhibitory binders to anti-TGF- β (TGF-b scFv VH-VL 1) showed superior efficacy to non-armored CAR-T, which induced 7 complete responses in 8 treated mice compared to the incomplete responses of control groups receiving an equivalent amount of non-armored CAR-T or non-transduced T cells. (FIG. 11).
Example 10 CAR-T cells secreting modulators of TGF-beta signaling enhance activation of host immune responses
This example demonstrates RNA Seq, showing that the host immune response is enhanced by activation by CAR-T cells secreting an anti-TGF- β binder.
At 0.2x10 6 Individual live EMT6-hCD19-Fluc tumor cells were inoculated into mammary fat pads (in situ) of 6-16 week old female Balb/c mice (Jackson Labs). After 6 days of implantation, tumor size reached about 50mm3 and mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm3; n=8 per group) and treated with cyclophosphamide (CPA; 200mg/kg i.p.). The next day, 2 million mouse CAR-T cells from the isotype CD45.1 Balb/c mice were injected into the tail vein. Group 1 received non-transduced control T cells, group 2 received non-armored CAR-T cells, group 3 received CAR-T cells secreting anti-TGF- β scFv VH-VL 1. Group 4 was treated with systemic anti-TGF-beta antibodies (clone 1D11.16.8; bioXcell;10mg/kg; 1.v weekly), and group 5 received isotype control antibodies (clone MOPC21; bioXcell;10mg/kg; 1.v weekly). Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) Length x width x heightx0.5236。
Mice were euthanized and tumors were harvested on day +12, snap frozen and kept at-80' c. RNA was extracted and RNA-Seq was performed and then analyzed computationally as shown in FIG. 12.
Tumor-infiltrating T cells (cd3d+, cd3e+, cd3g+) scores were significantly increased, particularly cd8+ T cells (cd8a+) and cell killing T cells (gzmb+), from tumors from mice receiving CAR-T secreting TGF- βscfv VH-VL1 compared to mice from other groups (fig. 13)
The ssGSEA enrichment score shows an increase in T cell characteristics and IFNg characteristics from tumors of mice receiving TGF-beta cFv VH-VL1 secreting CAR-T, indicating increased infiltration of the CAR-T cells and/or increased activation of the endogenous immune system. The increased characteristics of activated endothelial cells, co-stimulation and antigen presentation in tumors from mice receiving CAR-T secreting TGF- β scFv VH-VL1 clearly show activation of the endogenous immune system. Thus, armoring CAR-T cells with blocking antibodies (or other binders) inhibits TGF- β pathway antitumor efficacy, at least in part, by improving endogenous immune responses. (FIG. 14).
Example 11 FACS of tumor samples from EMT6-hCD19 mice treated with non-armored CAR-T cells
At 0.2x10 6 Individual live EMT6-hCD19-Fluc tumor cells were inoculated into mammary fat pads (in situ) of 6-16 week old female Balb/c mice (Jackson Labs). After 6 days of implantation, tumor size reached about 50mm3 and mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm3; n=8 per group) and treated with cyclophosphamide (CPA; 200mg/kg i.p.). The next day, 2 million mouse CAR-T cells from the isotype CD45.1 Balb/c mice were injected into the tail vein. Group 1 received non-transduced control T cells, group 2 received non-armored CAR-T cells, group 3 received CAR-T cells secreting anti-TGF-b scFv VH-VL1 monomer. Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) Length x width x height x0.5236.
Mice were euthanized and tumors were harvested on day +7, weighed and FACS analyzed. Briefly, tumors were cut into small pieces and digested using the mouse tumor dissociation kit (Miltenyi) according to the manufacturer's instructions. Samples were resuspended in PBS2% FCS, filtered and seeded into 96-well plates for FACS staining. Blocking Fc receptor (TruStain FcX (anti-mouse CD 16/32) antibody; biolegend) and labelling CAR with rhuCD19 (RnD Systems) at 4' C for 1 hour followed by labelling hCD19-Fc with anti-human IgG Fc antibody, surface markers including TCRa/b, CD8a, CD4, CD25, CD62L, CD b, gr1, CD11c, CD45.1 and CD45, staining of living cells with fixable vital dye (eBioscience) and intracellular antigens including GzmB, ki67 and FoxP3 were stained with the eBioscience Foxp 3/transcription factor staining buffer group (Thermofiser). Samples were filtered and taken on a BD Fortessa flow cytometer.
As shown in fig. 15, FACS staining showed a reduction in hcd19+ tumor cells and an increase in T cell infiltration (per mg of tumor tissue) in samples from mice receiving CAR-T secreting TGF- β scFv VH-VL compared to control groups receiving either non-transduced cells or non-armored CAR-T. Gating on CD45.1+ and CD45.1-T cells showed a particular increase in endogenous T cell infiltration (CD 45.1-). The level of CAR expression was higher for the transferred CAR-T cells (cd45.1+) from these samples, and cd8+ T cells showed higher CD25 expression, indicating increased activation. Higher GzmB expression from cd8+ T cells from host T cells (CD 45.1-) indicates higher cytotoxicity. Taken together, these FACS data indicate that armoring CAR-T cells with anti-TGF- β binders enhances the effect and endogenous immune response of CAR-T cells.
Example 12 xenograft models show human GCC-armoring with anti-TGF-beta or anti-TGF-beta R2 blocking antibodies Effect improvement of CAR-T cells
At 2x10 6 The individual live GSU tumor cells were inoculated subcutaneously into 6-16 week old female NSG mice (Jackson Labs). After 7 days of implantation, the tumor size reached about 50mm3 and the mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm3; n=6 per group). The following day, 500,000 or 100,000 human GCC CAR-T cells were injected into the tail And (3) veins. Group 1 received non-transduced control T cells, group 2 received non-armored CAR-T cells, group 3 received CAR-T cells secreting anti-TGF-b scFv VH-VL1 monomers, group 4 received CAR T cells secreting anti-tgfβr2vh3 monomers, and group 5 received CAR T cells secreting anti-tgfβr2vhh dimers. Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly and tumor volume was calculated using the following formula: tumor volume (mm) 3 ) Length x width x height x0.5236. GCC-CAR-T cells that were armored with anti-TGF- β or anti-tgfβr2 blocking antibodies showed a faster response at 500,000 cells than the unarmored control CAR-T and improved antitumor efficacy at 100,000 CAR-T cells. (FIG. 16).
Tumor and plasma concentrations of tgfβ modulators were determined using anti-Flag immunocapture LC/MS assays. As shown in fig. 17A-17D, small amounts of TGF- β antibodies or anti-TGFbR 2 antibodies were secreted in the circulation of mice treated with armored CAR-T cells. Plasma was collected from mice treated with a specified amount of armored or unarmored anti-GCC CAR-T cells using EDTA tubes.
As shown in fig. 20A-20C, the armored CAR-T cells also exhibited anti-tumor activity in GCC positive GSU, HT55, and MDA-MB-231-FP4 Luc xenograft models.
Liver metastasis was assessed using an intrasplenic injection of HT55 tumor cells followed by an intravenous injection of CAR-T cells. Armored CAR-T cells slowed metastasis to the liver relative to isotype control (fig. 21A-21C)
EXAMPLE 13 repeated antigen stimulation in GCC-positive tumors
100,000 anti-GCC CAR-T cells that are non-armored or armored (co-express anti-GCC CAR and tgfβ modulator (e.g., tgfβr2—vhh)) are co-cultured in duplicate with 200,000 HT29-GCC or HT29 parental (GCC negative) tumor cells in the presence or absence of TGF- β (1 ng/ml or 10 ng/ml). Half of the CAR-T cells per well were transferred to new tumor cell plates (with or without TGF- β 1ng/ml or 10 ng/ml) under the same conditions every 3-4 days. The supernatant was collected and frozen for later evaluation. Cell count and FACS staining analysis of cells were assessed.
Tumor cells were evaluated using CellTiterGlo (Promega) according to the manufacturer's protocol.
The plates were analyzed using a Pherastar reader. The percent kill was estimated using the following formula:
killing% = (1- (test well signal/control well signal)) =
Control wells contained tumor cells co-cultured with non-transduced T cells (for CAR-T cells) from the same donor. FACS staining was performed weekly using fluorochrome conjugated antibodies against human CD4, CD8, CD25 and the depletion markers PD-1, TIM-3, lag-3 and TIGIT antibodies (Biolegend). Dead cells were excluded using a fixable vital dye efluor 506 (thermofiser; according to manufacturer's protocol). CAR-expressing cells were incubated with GCC-hFc for 1 hour at 4'c, washed with PBS2% FCS and detected with secondary mouse anti-human IgG antibodies (30 min, 4' c).
After restimulation with target cells for several rounds (mimicking chronic antigen activation), TGF- β induces inhibition of CAR-T cell function. Only CAR-T cells that secrete tgfβ modulators (e.g., tgfβr2vhh dimer) are protected from inhibition by TGF- β (1 ng/ml or 10 ng/ml) stimulation (fig. 18A-18C). The inhibitory effect on CAR-T killing was correlated with proliferation and induced inhibition of the depletion marker Lag 3.
Example 14 repeated antigen stimulation in mesothelin (Msln) positive tumors
Approximately 100,000 iPSC-derived anti-Msln CAR-T cells (co-expressing an anti-Msln CAR with a tgfβ modulator (e.g., tgfβr2-VH or dnTGFbR 2)) or an anti-GFP control VH (Msln-control VH) were co-cultured in duplicate with 40,000 MiaPaca-2 tumor cells overexpressing human Msln in the presence or absence of TGF- β (R & D Systems,10 ng/ml). TGF beta R2-VH is secreted by CAR-T cells, while dnTGFbR2 binds to CAR-T cell membranes. Half of the CAR-T cells per well were transferred to new tumor cell plates (with or without TGF- β 10 ng/ml) under the same conditions every 3-4 days. The supernatant was collected and frozen for later evaluation. CAR-T cells were counted by flow cytometry at selected time points and FACS phenotyping was performed (fig. 22A).
Tumor cell viability was assessed using CellTiterGlo (Promega) according to the manufacturer's protocol. The plates were analyzed using a Pherastar reader. The percent kill was estimated using the following formula:
control wells contained tumor cells only but no effector (i.e., CAR-T) cells. The percent cytotoxicity is shown in figure 22B.
Dead cells were excluded for cell counting using a Sytox Red dye (thermosusher, according to manufacturer's protocol) and an equal volume of cell suspension was obtained using an HTS unit on a Fortessa flow cytometer (BD Biosciences). Viable CAR-T cells were counted by gating on living cells, single cells and size. The results were extrapolated to obtain the number of cells per well.
It was observed that TGF- β induced inhibition of CAR-T cell function (i.e., killing) and inhibited CAR-T cell proliferation after several rounds of restimulation with target cells (mimicking chronic antigen activation). Only CAR-T cells expressing tgfβ modulators (e.g., secreting tgfβr2vh dimer or expressing membrane bound dnTGFbR 2) are protected from inhibition by TGF- β (10 ng/ml), but not control VH.
Sequence listing
Table 5 below provides the descriptions and sequences disclosed herein.
TABLE 5 sequence listing
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Equivalent and scope
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the foregoing description, but is instead set forth in the following claims.
Having described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and the invention is described in detail by the appended claims.
Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which a method is performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
The articles "a" and "an" as used herein in the specification and claims should be understood to include plural referents unless the content clearly dictates otherwise. Unless otherwise indicated, if one, more than one, or all members of a group are present, used, or otherwise associated with a given product or process, the claims or descriptions including an "or" between one or more group members are deemed to be satisfied unless the context clearly indicates otherwise. The present invention includes embodiments in which exactly one member of the group is present, used, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one or the entire group member is present, utilized, or otherwise associated with a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations and permutations in which limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into the same basic claim (or any other claim concerned) in another dependent claim unless otherwise indicated or unless contradiction or inconsistency apparent to one of ordinary skill in the art. Where the elements are presented in a list (e.g., in Markush group (Markush group) or similar format), it is to be understood that each subgroup of the elements is also disclosed, and any element may be removed from the group. It should be understood that, in general, where the invention or aspects of the invention are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements, features, etc. For the sake of simplicity, these embodiments are not specifically set forth herein in every case in so many words. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a particular exclusion is set forth in the specification. Publications, web sites, and other reference materials cited to describe the background of the invention and provide additional details regarding its practice are hereby incorporated by reference.

Claims (20)

1. A population of genetically engineered T cells comprising a Chimeric Antigen Receptor (CAR) that recognizes a cancer-associated antigen and a tgfβ signaling pathway modulator.
2. The population of cells of claim 1, wherein the CAR recognizes an antigen selected from the group consisting of: ADGRE2, CLEC12, CAIX, CEA, CD, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, CEACAM 5, claudin 18.2, EGP-2, EGP-40, epCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY 2C), GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, leY, LI cell adhesion molecule, MAGE-AI, MUC1, MUC13, mesothelin, NKG2D ligand, NY-ES0-1, tumor embryo antigen (h 5T 4), PSCA, PSMA, PTK, ROR1, TAG-72, TROP2, VEGF-R2 and WT-1.
3. The population of cells of claim 1 or 2, wherein the tgfβ signaling pathway modulator binds to tgfβ or tgfβ receptor.
4. The population of cells according to any one of the preceding claims wherein said tgfβ signaling pathway modulator comprises an amino acid sequence selected from table 1.
5. The population of cells according to any one of the preceding claims wherein said CAR is a CD19 CAR or a GCC CAR.
6. The population of cells of claim 1 wherein the cells are autologous.
7. The population of cells of claim 1 wherein the cells are allogeneic.
8. The population of cells of any of the preceding claims, wherein the cells are genetically modified using a vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator.
9. The population of cells according to any of the preceding claims wherein the cells are genetically modified using two vectors, a first vector comprising a nucleic acid encoding a CAR polypeptide and a second vector comprising a nucleic acid encoding a tgfβ signaling pathway modulator.
10. The population of cells according to any preceding claim wherein said CAR comprises an intracellular signaling domain selected from the group consisting of: CD3 zeta chain, CD97, 2B4 GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, DAP10, DAP12, CD28 signaling domain, or combinations and variations thereof.
11. The population of cells according to any one of the preceding claims wherein said CAR comprises a transmembrane domain derived from a transmembrane domain selected from the group consisting of: CD3, CD8, CD28, OX40, CD27, 4-1BB, DAP10, DAP12, or combinations thereof.
12. A vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a tgfβ signaling pathway modulator.
13. The vector of claim 12, further comprising an internal ribosome entry site.
14. The vector of claim 12, further comprising a 2A self-cleavage site.
15. An immune cell modified with the vector of any one of claims 12-14.
16. The immune cell of claim 15, wherein the cell is a T cell.
17. A pharmaceutical composition comprising the population of immune cells of claim 1.
18. A method of modulating an immune response in a host, the method comprising administering the population of cells of claim 1 to the host, wherein the modulation of the immune response comprises one or more of the following by a host immune cell: increase ifnγ production; increase IL-2 production; increasing antigen presentation; and increase proliferation.
19. A method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the population of cells of claim 1.
20. The method of claim 19, wherein the cancer is selected from the group consisting of: leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, fahrenheit macroglobulinemia, heavy chain disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, solitary tumor, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, colorectal cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngeal tube tumor, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, and metastases thereof.
CN202280014920.1A 2021-02-15 2022-02-15 Cell therapy compositions and methods for modulating TGF-B signaling Pending CN117120077A (en)

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US63/149,628 2021-02-15
US202263306836P 2022-02-04 2022-02-04
US63/306,836 2022-02-04
PCT/IB2022/000063 WO2022172085A2 (en) 2021-02-15 2022-02-15 Cell therapy compositions and methods for modulating tgf-b signaling

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