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

Abstract

Methods of using polypeptides to modulate transforming growth factor-β (TGFβ) signaling (eg, TGFβ receptors, antibodies that specifically bind TGFβ or TGFβ receptors, or antigen-binding fragments thereof) are provided. Compositions comprising the antibodies or fragments thereof and methods of using the compositions to treat 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 invention also provides methods of treatment utilizing modulators of TGFβ signaling.

Description

Cell therapy compositions and methods for modulating TGF-B signaling

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/149,628 filed on February 15, 2021 and U.S. Provisional Application No. 63/306,836 filed on February 4, 2022, the disclosures of each of which are hereby incorporated by reference in their entirety.

Background Art

Immunotherapy using engineered cells targeting cancer-specific antigens has shown efficacy in the treatment of some cancers. However, malignant cells adapt to generate an immunosuppressive microenvironment to protect them from immune recognition and elimination. High TGFβ levels in the tumor microenvironment promote the maintenance and progression of some types of cancer cells. The tumor microenvironment poses a major challenge to therapeutic approaches involving the stimulation of immune responses, such as in the case of targeted cell therapy. Therefore, novel therapeutic strategies for the treatment of cancer are needed.

Summary of the invention

The present invention provides, in particular, a novel system for regulating TGFβ signaling for use in treating cancer (e.g., solid tumors). The present invention is based in part on the discovery that regulating transforming growth factor β (TGF-β) signaling can enhance adoptive cell therapy methods, such as targeted engineered chimeric antigen receptor (CAR) therapy. Regulating TGF-β signaling, such as by an antibody system described herein (e.g., anti-TGFβ or anti-TGFβR), an antigen binding fragment of TGF-βR2, or a recombinant extracellular domain, can alleviate the immunosuppressive microenvironment in the tumor and enhance the efficacy of immunotherapy.

T cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are designed to divert these efficient cells to the tumor microenvironment, where T cells can avoid negative regulatory signals and mediate effective tumor killing. Unwanted T cells can be eliminated by drug-induced dimerization of inducible caspase 9 with AP1903, indicating a method for pharmacologically activating a powerful switch that controls T cell populations (Di Stasi A et al., N Engl J Med. 2011; 365 (18): 1673-83). Therefore, although CARs can trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacle to the clinical application of this technology to date is that the in vivo expansion of CAR+T cells is limited, the cells disappear quickly after infusion, and the clinical activity is disappointing. Therefore, there is an urgent need in the art to find novel compositions and methods for treating cancer using methods that can show specific and effective anti-tumor effects without undesirable effects (i.e., high toxicity, insufficient efficacy).

The present invention addresses these needs by providing an immunomodulatory system comprising a CAR and a TGFβ signaling pathway regulator expressed in an immune cell (e.g., a T cell). Compositions and treatment methods comprising the immunomodulatory system can be used to treat cancer and other diseases and/or disorders. In particular, the present invention provides engineered immune cells expressing armored CARs, which can be used to treat diseases, disorders, or disorders (e.g., cancer, solid tumors) associated with dysregulated expression of TGFβ. Armored CAR T cells co-expressing TGFβ regulators show high surface expression of CAR on transduced T cells, and enhanced cell lysis of cancer cells. Therefore, the present invention provides methods and compositions for enhancing immune responses to cancer and pathogens using an immunomodulatory system (e.g., engineered CAR T cells) comprising a polypeptide that regulates TGF-b signaling.

The present invention provides, in part, improved CAR polypeptides comprising TGFβ signaling pathway regulators, nucleic acid molecules encoding such polypeptides, cells (e.g., T cells) genetically modified to express the improved CAR, and methods of using the modified cells in adoptive cell therapy for treating cancer (e.g., solid tumor cancer).

In some embodiments, the present invention provides CAR-T cells modified to express a TGFβ signaling pathway regulator (also referred to herein as "TGFβ 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 TGFβ signaling pathway regulator (also referred to herein as "unarmored CAR-T cells").

In some aspects, the present invention provides immunoreactive cells (e.g., T cells) with antigen receptors, which may be chimeric antigen receptors (CARs), and which include polypeptides that regulate TGF-b signaling. These engineered immunoreactive cells (e.g., CAR-T cells) are directed against antigens and can resist immunosuppression and/or have enhanced immune activation properties.

In one aspect, the present invention provides a genetically engineered T cell population comprising a chimeric antigen receptor (CAR) that recognizes a cancer associated antigen and a TGFβ signaling pathway regulator.

In some embodiments, the cell population comprises a CAR that recognizes an antigen selected from the group consisting of ADGRE2, CLEC12, CAIX, CEA, CD5, 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 proteins (Claudin) 18.2, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY2C), GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, LeY, LI cell adhesion molecules, MAGE-AI, MUC1, MUC13, mesothelin, NKG2D ligand, NY-ES0-1, tumor embryonic antigen (h5T4), PSCA, PSMA, PTK7, ROR1, TAG-72, TROP2, VEGF-R2 and WT-1.

In some embodiments, the cell population 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 regulator comprising an amino acid sequence selected from Table 1.

In some embodiments, the cell population is autologous.

In some embodiments, the cell population is allogeneic.

In some embodiments, the cell population is a primary cell. In some embodiments, the cell population is derived from an induced pluripotent stem cell (iPSC).

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 regulator.

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 regulator.

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-retrovirus), lentiviral transduction, transposons and transposases (Sleeping 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 cell population comprises a CAR comprising a transmembrane domain, wherein the transmembrane domain is derived from a transmembrane domain selected from the group consisting of: CD3, CD8, CD28, OX40, CD27, 4-1BB, DAP10, DAP12, or a combination thereof.

In one aspect, the present invention provides a vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a TGFβ signaling pathway regulator.

In some embodiments, the vector comprising a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a TGFβ signaling pathway regulator comprises an internal ribosome entry site.

In some embodiments, the vector further comprises a 2A ribosomal sequence.

In one aspect, the present invention provides an immune cell modified with a vector, wherein the vector comprises a first nucleic acid encoding a CAR polypeptide and a second nucleic acid encoding a TGFβ signaling pathway regulator.

In some embodiments, the immune cell is a T cell.

In one aspect, the present invention provides a method for regulating an immune response in a host, the method comprising administering to the host a population of genetically engineered T cells, the cell population comprising a chimeric antigen receptor (CAR) that recognizes a cancer-associated antigen and a TGFβ signaling pathway regulator, wherein the regulation of the immune response comprises one or more of the following by the host immune cells: increasing IFNγ production; increasing IL-2 production; increasing antigen presentation; and increasing proliferation.

In one aspect, the present invention provides a pharmaceutical composition comprising a genetically engineered T cell population, wherein the cell population comprises a chimeric antigen receptor (CAR) that recognizes a cancer-associated antigen and a TGFβ signaling pathway regulator.

In one aspect, the present invention provides a method for treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a genetically engineered T cell population, the cell population comprising a chimeric antigen receptor (CAR) that recognizes a cancer-associated antigen and a TGFβ signaling pathway regulator.

In some embodiments, the cancer is selected from the group consisting of leukemia, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, carcinomas, fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcomas, osteogenic sarcomas, chordoma, angiosarcomas, endotheliosarcomas, lymphangiosarcomas, lymphangioendotheliosarcomas, synovioma, mesothelioma, Ewing's tumor, 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 carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, colorectal cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma and their metastases.

In one aspect, the present invention provides an immunomodulatory system comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a nucleic acid sequence encoding a polypeptide that modulates TGF-b signaling (e.g., a TGFβ signaling regulator).

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 (sdAb), camelized antibodies; masked antibodies, small modular immunopharmaceuticals ("SMIPsTM"), single chains, tandem bivalent antibodies, VHH, Anticalins, nanobodies, humanized antibodies (humabodies), minibodies, BiTEs, ankyrin repeat proteins, DARPINs, Avimers, DARTs, TCR-like antibodies, Adnectins, Affilins, Trans-bodies; Affibodies, TrimerX, miniproteins, Fynomers, Centyrins; and KALBITOR, or fragments 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 binding agent.

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-bR2).

In some embodiments, the polypeptide that modulates TGF-b signaling comprises TGF-b receptor 2 (TGF-bR2) or a fragment thereof.

In some embodiments, the polypeptide that modulates TGF-b signaling comprises the extracellular domain of TGF-bR2 (TGF-bR2).

In some embodiments, the CAR binds to an antigen selected from the group consisting of: ADGRE2, CLEC12, CAIX, CEA, CD5, 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 GUCY2C), 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 embryonic antigen (h5T4), PSCA, PSMA, PTK7, 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 immunomodulatory system according to any of the preceding claims, wherein 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 a combination 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 a basic-rich stretch (BRS) region, wherein the BRS region is BRS1, BRS2, and BRS3.

In one aspect, the present invention provides a nucleic acid comprising the immune modulation system described herein, wherein the sequence encoding the chimeric antigen receptor (CAR) and the sequence encoding the polypeptide that 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 the immune modulatory system 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 the immunomodulatory system described herein.

In one aspect, the present invention provides an immunoreactive cell comprising: a targeting agent specific for a tumor-associated antigen or a stress ligand, and a nucleic acid encoding a recombinant polypeptide that regulates 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 present invention provides an immunoresponsive cell comprising: a chimeric antigen receptor (CAR); and a nucleic acid encoding a recombinant polypeptide that regulates TGF-b signaling.

In some embodiments, the CAR and the nucleic acid encoding a polypeptide that modulates TGF-b signaling are located 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 binding agent.

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-bR2) or a fragment thereof.

In some embodiments, the recombinant polypeptide that modulates TGF-b signaling comprises the extracellular domain of TGF-bR2.

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-bR2).

In some embodiments, the immunoreactive cell comprises a CAR expressed by a vector, an engineered mRNA, or integrated into a host cell chromosome. In some embodiments, the sequence encoding the CAR 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 Cas13.

In some embodiments, the immunoreactive cells comprise a recombinant polypeptide that modulates TGF-b signaling, which is expressed from a vector, engineered mRNA, or integrated into a host cell chromosome. In some embodiments, the sequence encoding the polypeptide that modulates TGF-b signaling is integrated into a host cell chromosome using Crispr/Cas9, Cas12a, or Cas13.

In some embodiments, the immunoreactive cells are selected from the group consisting of: T cells, natural killer (NK) 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 can 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, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, CEACAM5, tight junction protein 18.2, EGP-2, EGP-40, E pCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY2C), 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 embryonic antigen (h5T4), PSCA, PSMA, PTK7, 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 CD3ζ, 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 a combination 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 a basic-rich stretch (BRS) region, wherein the BRS region is BRS1, BRS2, and BRS3.

In some embodiments, the immunoreactive cell comprises 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 present invention provides a pharmaceutical composition comprising an effective amount of the immunomodulatory system described herein.

In one aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a nucleic acid sequence encoding the immune regulatory system described herein.

In one aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a vector encoding the immunomodulatory system described herein.

In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of the immunoreactive cells described herein.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In one aspect, the present invention provides a kit for treating cancer, the kit comprising an immunoreactive cell comprising a chimeric antigen receptor (CAR); and a nucleic acid encoding a recombinant polypeptide that regulates TGF-b signaling.

In some embodiments, the kit comprises a nucleic acid or vector encoding an immunomodulatory system described herein.

In one aspect, the present invention provides a method for treating or preventing cancer or its metastasis in a subject, the method comprising administering an effective amount of an immunoreactive cell comprising a chimeric antigen receptor (CAR); and a nucleic acid encoding a recombinant polypeptide that regulates TGF-b signaling.

In some embodiments, the compositions described herein can be used to treat 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 leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, carcinomas, fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma 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 carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, colorectal cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

In some embodiments, the second therapeutic agent is administered to the subject systemically.

In some embodiments, the second therapeutic agent is administered separately from the CAR and the nucleic acid encoding the recombinant polypeptide that modulates TGF-β 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 present invention provides a method for regulating immune cell activity, the method comprising administering a nucleic acid encoding a chimeric antigen receptor (CAR); and a nucleic acid encoding a recombinant polypeptide that regulates TGF-b signaling.

In one aspect, the present invention provides a method for modulating the activity of a chimeric antigen receptor (CAR), the method comprising administering a nucleic acid encoding a chimeric antigen receptor (CAR); and a nucleic acid encoding a recombinant polypeptide that modulates 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 immunomodulatory system comprising a nucleic acid, vector or immunoreactive cell 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 eradicates the tumor in the subject.

In one aspect, the invention provides a method of increasing production of immune-activating cytokines in response to cancer cells in a subject, the method comprising administering to the subject an immunomodulatory system comprising a nucleic acid, vector or immunoreactive cell described herein.

In one aspect, the present invention provides a method for producing antigen-specific immunoreactive cells, the method comprising introducing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a recombinant polypeptide that regulates TGF-b signaling into immunoreactive cells.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings included herein, which consist of the following figures, are for illustration purposes only and are not limiting.

Figures 1A-1E show exemplary expression of CAR and TGF-β signaling regulators in immunoreactive cells (e.g., transduced T cells). Figure 1A illustrates a lymphocyte population, Figure 1B illustrates a single cell population, Figure 1C depicts a live CD3+ cell population, and Figure 1D shows exemplary flow cytometry results evaluating CAR expression in armored human CAR-T cells expressing TGF-β. FIG. 1E depicts a histogram showing the transduction efficiency as the percentage of live cells positive for CAR staining using unarmored cells transduced with CD19 CAR alone, and CD19 CAR-T cells armored with TGFbscFv 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), mTGFbR2VH1 (SEQ ID NO: 6) and hTGFbR2 VH1 (SEQ ID NO: 8), or untransduced cells.

Figures 2A-2B are graphs showing exemplary in vivo killing assay results of CD19+ Raji cells (Figure 2A) and CD19ko Raji cells (Figure 2B) by immunoreactive cells co-expressing anti-CD19 CAR and TGF-β signaling regulators, using effectors relative to targets of: unarmored cells transduced with CD19 CAR alone, and CD19 armored with TGFbscFv 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), mTGFbR2VH1 (SEQ ID NO: 6), and hTGFbR2 VH1 (SEQ ID NO: 8) CAR-T cells, or untransduced cells.

FIG3A shows a histogram illustrating exemplary ELISA results showing secretion of TGF-β binders by human CAR-T cells, and FIG3B shows a histogram illustrating exemplary ELISA results showing secretion of TGFβR2 binders by human CAR-T cells, as well as their ability to bind to cognate antigens.

FIG4 depicts a histogram depicting exemplary luciferase assay results evaluating the inhibition of TGF-β signaling by supernatants from CAR-T cells secreting 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).

The histogram shown in Figure 5A depicts the results of an exemplary luciferase assay evaluating the inhibition of TGF-β signaling by supernatants from CAR-T cells secreting TGFb-scFv VH-VL1 G4S dimer (SEQ ID NO: 17), TGFb-scFv VH-VL1 2xG4S dimer (SEQ ID NO: 18), TGFb-scFv VH-VL1 miniantibody (SEQ ID NO: 21), TGFb-scFv VH-VL1 miniantibody + hinge (SEQID NO: 19). Figure 5B shows a schematic diagram of an exemplary TGF-β modulator, which is designed and screened for secretion of multimeric binders for TGF-β using a luciferase reporter assay. Figure 5C shows a schematic diagram of an exemplary TGF-β modulator comprising a VHH binding domain.

The histogram shown in Figure 6A depicts 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-scFvVH-VL1 G4S dimer (SEQ ID NO: 17) binders compared to unarmored cells expressing CAR alone. Figure 6B shows a histogram depicting exemplary luciferase assay results evaluating the relative blocking activity of TGFβR2VHH and scFv monomer and dimer constructs. Unarmored CAR-T cells, mTGFbR2 VH2 monomer, mTGFbR2 VH2 G4S dimer, mTGFbR2 VH2 G4S trimer, hTGFbR2 VH2 monomer, hTGFbR2 VH2 G4S dimer, hTGFbR2 VH3 monomer, hTGFbR2 VH3 G4S dimer, hTGFbR2scFv VH-VL monomer, hTGFbR2 scFv VH-VL G4S dimer.

The histograms depicted in Figures 7A and 7B illustrate exemplary ELISA results, showing that exemplary TGFb modulators bind to human TGFbR2 (Figure 7A) but not to mouse TGFbR2 (Figure 7B). Unarmored CAR-T cells, mTGFbR2 VH2 monomer, mTGFbR2VH2 G4S dimer, mTGFbR2 VH2 G4S trimer, hTGFbR2 VH2 monomer, hTGFbR2 VH2 G4S dimer, hTGFbR2 VH3 monomer, hTGFbR2 VH3 G4S dimer, hTGFbR2 scFv VH-VL monomer, hTGFbR2scFv VH-VL G4S dimer.

Figure 8A shows an exemplary injection schedule to evaluate tumor growth of EMT6-hCD19-Fluc tumor cells as described in Example 6. Figure 8B shows exemplary tumor volume changes over time in mice receiving CAR-T cells that secrete TGF-β binders relative to unarmored CAR-T or untransduced CAR-T cells. Figure 8C shows exemplary liver metastases in mice treated with CAR-T cells that secrete TGF-β binders relative to unarmored or untransduced CAR-T cells. Figure 8D shows exemplary lung metastases in mice treated with CAR-T cells that secrete TGF-β binders relative to unarmored or untransduced CAR-T cells. Figure 8E shows exemplary imaging results of tumor cells expressing luciferase in liver and lung tissues.

Figures 9A and 9B show histograms comparing exemplary SBE-Luc TGF-b reporter assay results from armored mouse CAR-T cells secreting different TGF-b ligand traps (TGF-b scFvVH-VL1 to TGFbR2 ECD monomers, homodimers (Figure 9A), and heterodimers (Figure 9B)) with supernatants from unarmored CAR-T cells.

Figure 10A shows an exemplary injection schedule to evaluate the tumor growth of EMT6-hCD19-Fluc tumor cells. Figure 10B shows the changes in exemplary tumor volume over time in mice receiving untransduced T cells or unarmored CAR-T cells (CAR-T cells that do not co-express TGFβ signaling regulators). Figure 10C shows the changes in exemplary tumor volume over time in mice receiving armored CAR-T cells or unarmored CAR-T cells (CAR-T cells that do not co-express TGFβ signaling regulators) that co-express TGFbR1+2ECD dimers. Figure 10D shows the changes in exemplary tumor volume over time in mice receiving systemic anti-TGFb antibodies (1D11) or unarmored CAR-T cells (CAR-T cells that do not co-express TGFβ signaling regulators).

Figure 11 depicts a graph showing exemplary tumor volume changes over time in mice developed from MC38 cells expressing CD19. Mice received untransduced T cells, unarmored anti-CD19 CAR-T cells, or CAR-T cells secreting an inhibitory binder against TGF-b (TGF-b scFv VH-VL1).

FIG. 12 depicts a graph showing an exemplary RNA Seq analysis demonstrating enhanced activation of host immune responses by CAR-T cells secreting an anti-TGF-b (TGF-b scFvVH-VL1 (SEQ ID NO: 1)) binder.

Figure 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).

Figure 14 shows an exemplary single sample gene set enrichment analysis (GSEA), with enrichment scores demonstrating increased T cell and IFNg signatures in tumors of mice receiving CAR-T cells secreting TGF-bscFv VH-VL1 (SEQ ID NO: 1).

Figure 15 shows an exemplary surface marker analysis including TCRa/b, CD8a, CD4, CD25, CD62L, CD11b, Gr1, CD11c, CD45.1, and CD45 in mice receiving untransduced control T cells, unarmored CD19 CAR-T cells, or CAR-T cells secreting anti-TGF-b scFv VH-VL1 (SEQ ID NO: 1) monomers.

16A and 16B are graphs depicting exemplary in vivo analyses of a GSU xenograft model demonstrating improved function of human GCC-CAR-T cells armored with anti-TGF-b or anti-TGFbR2 blocking antibodies.

Figures 17A to 17D show tumor and/or plasma concentrations of TGFb modulators secreted by anti-GCC CAR-T cells co-expressing TGF-b scFv VH-VL1 and TGFbR2VHH, as determined using anti-Flag immunocapture LC/MS assay.

Figures 18A to 18C are graphs depicting exemplary in vitro killing assay results in HT29-GCC positive cells using unarmored anti-GCC CAR-T cells, anti-TGFbR2 VHH monomer armored anti-GCC CAR-T cells, and anti-TGFbR2 VHH dimer armored anti-GCC CAR-T cells in the absence of TGFb (Figure 18A) and in the presence of TGFb (Figure 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.

Figures 20A to 20C depict xenograft models of GCC-expressing cells GSU (Figure 20A), HT55 (Figure 20B), and MDA-MB-231-FP4 Luc (Figure 20C) treated with armored and unarmored CAR-T cells and CAR-T cells expressing dominant negative TGFbR2 (dnTGFbR2).

Figures 21A-21C show exemplary results of the HT55 liver metastasis model treated with armored anti-GCC CAR T cells.

Figure 22A shows CAR-T cells counted by flow cytometry and FACS phenotypes performed at designated time points. Figure 22B shows the percentage of cytotoxicity in anti-Msln CAR-T cells, which co-express CARs against Msln together with TGFβ regulators (e.g., TGFβR2-VH or dnTGFbR2), or CARs against GFP (Msln-control VH) together with control VH.

definition

To make the present 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 which will become apparent to those skilled in the art upon reading this disclosure, and so forth.

Administration: As used herein, "administering" a composition to a subject means providing, applying, or contacting the composition with the subject. Administration can be achieved 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 terms "adoptive cell therapy" or "adoptive cell transfer" or "cell therapy" or "ACT" refer to the transfer of cells (e.g., a population of genetically modified cells described herein) to patients in need. The cells may be derived and propagated from patients in need (i.e., autologous cells), or may be obtained from non-patient donors (i.e., allogeneic cells). In some embodiments, the cells are immune cells, such as lymphocytes, modified to express CAR and TGFβ signaling pathway regulators, as described herein (e.g., TGFβ armored CAR-T cells). Various cell types may be used for ACT, including but not limited to natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, γδT cells, regulatory T cells, induced pluripotent stem cells (iPSC), iPSC-derived T cells, iPSC-derived NK cells, hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and peripheral blood mononuclear cells.

Affinity: As used herein, the term "affinity" refers to the properties of a binding interaction between a binding moiety (e.g., an antigen binding agent (e.g., a variable domain described herein) and a target (e.g., an antigen (e.g., TGFΒ or TGFBR)), and is indicative of the strength of the binding interaction. In some embodiments, the measurement of affinity is expressed as a dissociation constant ( _KD ). The binding affinity of an antigen binding protein to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), kinetics (e.g., BIACORE ^™ analysis), or other methods known in the art.

Avidity: As used herein, the term "avidity" is the sum of the strengths with which two molecules bind to each other at multiple sites (eg, taking into account the valency of the interaction).

Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a 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, animals may be transgenic animals, genetically engineered animals, and/or clones.

Autologous: As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

Allogeneic: 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. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may differ genetically enough to allow antigenic interaction.

Antibody or antigen-binding agent: As used herein, the term "antibody" or "antigen-binding agent" refers to a polypeptide comprising a typical immunoglobulin sequence element sufficient to confer specific binding to a specific target antigen. Those skilled in the art will appreciate that the terms can be used interchangeably herein. In some embodiments, as used herein, the term "antibody" or "antigen-binding agent" also refers to an "antibody fragment" or "multiple antibody fragments" comprising a portion of a complete 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; tri-antibodies; tetra-antibodies; linear antibodies; single-chain antibody molecules; and CDR-containing portions included in multispecific antibodies formed by antibody fragments. Those skilled in the art will appreciate that the term "antibody fragment" does not imply and is not limited to any particular mode of production. Antibody fragments can be prepared using any appropriate method, including but not limited to cleavage, chemical synthesis, recombinant production, and the like of complete antibodies. As is known in the art, naturally occurring intact antibodies are tetrameric agents of approximately 150 kD, comprising two identical heavy chain polypeptides (each approximately 50 kD) and two identical light chain polypeptides (each approximately 25 kD) bound to each other to form a so-called "Y-shaped" structure. Each heavy chain comprises at least four domains (each approximately 110 amino acids long), an amino-terminal variable ( _VH ) domain (located at the top of the Y structure), followed by three constant domains: _CH1 , _CH2 , and a carboxy-terminal _CH3 (located at the bottom of the Y-shaped backbone). A short region (called a "switch") connects the heavy chain variable and constant regions. A "hinge" connects the _CH2 and _CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides in the intact antibody to each other. Each light chain comprises two domains, an amino-terminal variable ( _VL ) domain, followed by a carboxy-terminal constant ( _CL ) domain, separated from each other by another "switch". A complete antibody tetramer consists of two heavy chain-light chain dimers, in which the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds link the heavy chain hinge regions to each other, so that the dimers are linked to each other and form a tetramer. Naturally produced antibodies are also glycosylated, usually on the _CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold", which consists of two β-sheets (e.g., 3-, 4-, or 5-chain sheets) stacked against each other in a compressed antiparallel β barrel. Each variable domain contains three hypervariable loops, called "complementarity determining regions" (CDR1, CDR2, and CDR3) and four slightly invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form a β-sheet, providing a structural framework for the domains, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space, so that they produce a single hypervariable antigen binding site located at the top of the Y structure. Amino acid sequence comparisons between antibody polypeptide chains have defined two light chain (κ and λ) classes, several heavy chain (e.g., μ, γ, α, ε, δ) classes, and certain heavy chain subclasses (α1, α2, γ1, γ2, γ3, and γ4). Antibody classes (IgA [including IgA1, IgA2], IgD, IgE, IgG [including IgG1, IgG2, IgG3, and IgG4], and IgM) are defined based on the class of the heavy chain sequence used.

For the purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex including sufficient immunoglobulin domain sequences found in natural antibodies may be referred to and/or used as an "antibody" or "antigen-binding agent", regardless of whether such polypeptides are naturally produced (e.g., produced by an organism that responds to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. 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 mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, antibody sequence elements are humanized, primatized, chimeric, etc. In addition, as used herein, the term "antibody" or "antigen-binding agent" will be understood to cover (unless otherwise specified or obvious from the context) in appropriate embodiments, it may refer to 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 affinity, etc.) antibodies, small modular immunopharmaceuticals ("SMIPs ^TM "), single chain antibodies, camelized antibodies, and/or antibody fragments. In some embodiments, an antibody may lack covalent modifications (e.g., attached glycans) that it may have when produced naturally. In some embodiments, an antibody may contain covalent modifications (e.g., attached glycans, payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.], or other side groups [e.g., polyethylene glycol, etc.]).

Approximately or approximately: As used herein, the term "approximately" or "approximately" as applied to one or more values of interest refers to a numerical value similar to the stated reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term "approximately" or "approximately" refers to falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a smaller numerical range (but this numerical value will exceed 100% of the possible value) in either direction. It should be understood that when the term "approximately" or "approximately" is used to modify the stated reference value, the stated reference value itself and the numerical value of the reference value on either side of the stated reference value are covered.

Armored CAR-T cells: As used herein, the term "armored CAR cells" or "armored CAR-T cells" refers to genetically engineered cells that have the ability to evade tumor immunosuppression and tumor-induced CAR-T dysfunction. In some embodiments, the armored CAR T cells comprise a chimeric antigen receptor (CAR) that recognizes a cancer-associated antigen and a TGFβ signaling pathway regulator.

Complementarity Determining Region (CDR): A "CDR" of a variable domain is an amino acid residue within the variable region that is identified according to the definitions of Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact and/or conformational definitions, or any CDR determination method well known in the art. Antibody CDRs can be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, NIH, Washington DC. The positions of CDRs can also be identified as structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other CDR identification methods include the "AbM definition," which is a compromise between Kabat and Chothia and is a method developed using Oxford Molecular's AbM antibody simulation software (now ) derived from, or based on the "contact definition" of CDRs of observed antigen contacts, as described in MacCallum et al., J. Mol. Biol., 262: 732-745, 1996. In another method referred to herein as the "conformational definition" of CDRs, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008. There are other CDR boundary definitions that 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 be shortened or extended, or even the entire CDR will not significantly affect antigen binding, based on predictions or experimental results of specific residues or residue groups. As used herein, CDRs may refer to CDRs defined by any method known in the art (including combinations of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the 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 target cells carrying the antigen and subsequently kill the target cells with cytotoxins. The antibodies "arm" the cytotoxic cells and are necessary for killing target cells by this mechanism. The main cells that mediate ADCC are NK cells, which express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). In order to assess the ADCC activity of the molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No. 5,500,362 or No. 5,821,337. Available effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, such as in an animal model, such as in Clynes et al., PNAS USA 95: In an animal model disclosed in 652-656 (1998).

Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response; and/or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell) when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies); alternatively or additionally, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). One 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), rather than in all members of the target organism species. In some embodiments, the antigen triggers 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 antibodies and/or T cell receptors and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to antibodies and/or T cell receptors in vitro, regardless of whether this interaction occurs in vivo. In some embodiments, the antigen reacts with the product of a specific humoral or cellular immunity, including those induced by a heterologous immunogen.

Associated: As the term is used herein, two events or entities are "associated" with each other if one of them is associated with the presence, level, and/or form of the other. For example, if the presence, level, and/or form of a particular entity (e.g., a polypeptide) is associated with the incidence and/or susceptibility of a particular disease, disorder, or condition (e.g., in a related population), the particular entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly so that they are physically close to each other and remain physically close. In some embodiments, two or more entities that are physically associated with each other are covalently attached to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently attached to each other, but are associated in a non-covalent form, such as by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, when referring to "antigens associated with cancer cells," the term "associated with..." refers to the presence of specific antigens on the surface of cancer cells.

Binding: It should be understood that the term "binding," as used herein, generally refers to a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts, including the interacting entities or moieties in isolation or in the context of a more complex system (e.g., covalently or otherwise associated with a carrier entity and/or in a biological system or cell). As used herein, "K _a " refers to the rate of association of a specific binding moiety with a target to form a binding moiety/target complex. As used herein, "K _d " refers to the rate of dissociation of a specific binding moiety/target complex. As used herein, "K _D " refers to the dissociation constant, which is derived from the ratio of K _d to _Ka (i.e., K _d /K _a ), and is expressed in molar concentration (M). K _D values can be measured using methods well established in the art (e.g., by using surface plasmon resonance) or using a biosensor system (e.g. system) to measure.

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 oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or includes one or more solid components.

Characteristic portion: As used herein, the term "characteristic portion" is used in the broadest sense to refer to a portion of a substance whose presence (or absence) is associated with the presence (or absence) of a particular characteristic, property, or activity. In some embodiments, a characteristic portion of a substance is a portion found in the substance and related substances that share a particular characteristic, property, or activity, but not those that do not share the particular characteristic, property, or activity.

Chimeric Antigen Receptor: As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor consisting 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 desired cell surface antigens or MHC-peptide complexes. These synthetic receptors typically contain a target binding domain associated with one or more signaling domains via a flexible linker in a single fusion molecule. The target binding domain is used to guide immune cells (e.g., T cells) to specific targets on the surface of pathological cells (e.g., cancer cells), and the signaling domain contains molecular mechanisms for activation and proliferation of immune cells (e.g., T cells). A 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 CAR. CAR has successfully redirected immune cells (T cells) to antigens expressed on the surface of tumor cells from various malignant diseases (including lymphomas and solid tumors) (Gross et al., (1989) Transplant Proc., 21 (1 Pt 1): 127-30; Jena et al., (2010) Blood, 116 (7): 1035-44). The extracellular binding domain of CAR can be composed of a single-chain variable fragment (scFv) derived from the fusion of the variable heavy chain and light chain regions of a murine or humanized monoclonal antibody. In some embodiments, the extracellular binding domain comprises a single domain antibody. Alternatively, a scFv derived from Fab (rather than an antibody derived from, for example, a Fab library) can be used. In multiple embodiments, this scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain.

At least three generations of CAR have been developed. The first generation of CAR includes a target binding domain attached to a signaling domain of a cytoplasmic region derived from CD3ζ or Fc receptor γ chain. It has been shown that the first generation of CAR successfully redirects T cells to selected targets, but it fails to provide long-term amplification and anti-tumor activity in vivo. The second and third generation CARs focus on enhancing the survival rate of modified T cells and increasing proliferation by including co-stimulatory molecules such as CD28, OX-40 (CD134) and 4-1BB (CD137). The embodiments described herein are partially focused on further improving immunotherapy containing CAR-T, for example, by armoring CAR-T with TGFβ signaling pathway regulators, thereby making immunotherapy more effective in treating cancer (especially solid tumor cancer). Relative to unarmored CAR-T cells, the armored CAR provided herein can improve or enhance CAR-T function and survival rate when facing an unfavorable tumor microenvironment.

Codon optimization: As used herein, a "codon optimized" nucleic acid sequence refers to a nucleic acid sequence that has been altered so that translation of the nucleic acid sequence and expression of the resulting protein is improved and optimized for a specific expression system. A "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, a nucleic acid sequence can be "codon optimized" to be expressed 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.

Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., which may be different from each other but are sufficiently similar to allow comparisons to be made between them so that conclusions can reasonably be drawn based on the observed differences or similarities. One of ordinary skill in the art will understand what degree of identity is required in any given case in the context in order for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.

Corresponding to: As used herein, the term "corresponding to" is generally used to specify the position/identity of an amino acid residue of a polypeptide of interest. One of ordinary skill in the art will understand that, for simplicity, residues in a polypeptide are generally named using a canonical numbering system based on a reference related polypeptide, thus, an amino acid "corresponding to" residue position 190, for example, is not actually necessarily the 190th amino acid in a particular amino acid chain, but rather corresponds to the 190th residue in the reference polypeptide; one of ordinary skill in the art readily understands how to identify a "corresponding" amino acid.

Derived from: As used herein, the phrase "derived from" or "specific for a specified sequence" refers to a sequence that contains 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 continuous region of, for example, a specified sequence. In certain embodiments, the sequence comprises all of the specified nucleotide or amino acid sequence. As determined by techniques known in the art, the sequence may be complementary (in the case of a polynucleotide sequence) or identical to a sequence region that is unique to a specific sequence. Regions from which sequences may be derived include, but are not limited to, regions encoding specific epitopes, regions encoding CDRs, regions encoding framework sequences, regions encoding constant domain regions, regions encoding variable domain regions, and untranslated and/or untranscribed regions. The derived sequence is not necessarily physically derived from the sequence of interest under study, but may be produced 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. Therefore, it can represent the synonymous or antisense direction of the original polynucleotide. In addition, the regional combination corresponding to the specified sequence can be modified or combined in a manner known in the art to meet the intended use. For example, a sequence may include two or more continuous sequences, each of which includes a part of the specified sequence, and is interrupted by a region different from the specified sequence but intended to represent a sequence derived from the specified sequence. With regard to antibody molecules, "derived from" includes antibody molecules functionally or structurally related to the comparison antibody, for example, "derived from" includes antibody molecules with similar or substantially identical sequences or structures, such as antibody molecules with identical or similar CDRs, frameworks or variable regions. "Derived from" of an antibody also includes residues, such as one or more, such as 2,3,4,5,6 or more residues, which may be continuous or discontinuous, but defined or identified according to a numbering scheme or with the homology of the general antibody structure or three-dimensional proximity (i.e., in CDR or framework regions) of the comparison sequence. The term "derived from" is not limited to physical derivation, but includes generation by any means, such as by using sequence information from a comparison antibody to design another antibody.

Determination: Many of the methods described herein include a "determination" step. Those of ordinary skill in the art who read this specification will understand that this "determination" can be performed using any of a variety of techniques available to those skilled in the art, including, for example, specific techniques explicitly mentioned herein. In some embodiments, determination involves manipulation of physical samples. In some embodiments, determination involves consideration and/or manipulation of data or information, such as using a computer or other processing unit suitable for performing relevant analysis. In some embodiments, determination involves receiving relevant information and/or materials from a source. In some embodiments, determination involves comparing one or more features of a sample or entity with a comparable reference.

Engineered: As used herein, the term "engineered" describes a polynucleotide, polypeptide, or cell that has been designed or modified by man and/or whose existence and production requires human intervention and/or activity. For example, an engineered cell that is designed to elicit a specific effect that is different from the effect of a naturally occurring cell of the same type. In some embodiments, the engineered cell expresses a chimeric antigen receptor described herein.

Effector function: As used herein, the term "effector function" refers to a biological activity attributable to an antigen binding agent described herein. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; and B cell activation). "Reducing or minimizing" antibody effector function means 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. The determination of antibody effector function can be easily determined and measured by a person of ordinary skill in the art. In some embodiments, the antibody effector functions of complement binding, complement dependent cytotoxicity, and antibody-dependent cellular toxicity are affected. In some embodiments, the effector function is eliminated via a mutation that eliminates glycosylation in the constant region, such as a "no effector mutation". In one aspect, the effector-free mutation is N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively, additional mutations that result in reduced or eliminated effector function include: K322A and L234A/L235A (LALA). Alternatively, effector function can be reduced or eliminated by production techniques, such as expression in a glycosylation-free host cell (e.g., E. coli), or wherein the glycosylation pattern is altered 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., an antibody or receptor) binding component. In some embodiments, an epitope consists of a plurality of amino acids in an antigen. In some embodiments, such amino acid residues are exposed to the surface when the antigen adopts a relevant three-dimensional conformation. In some embodiments, when the antigen adopts such a conformation, the amino acid residues are physically close to or equal in height 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.

Excipients: As used herein, the term "excipient" refers to non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilization effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

Expression: The term "expression" or "expressed", when used in reference to a nucleic acid herein, refers to one or more of the following events: (1) production of an RNA transcript from a 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 a process in which cells are removed from a living organism and propagated outside of the organism (eg, 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 nucleic acid sequences encoding at least a portion of two different (e.g., heterologous) proteins. The skilled artisan will no doubt be aware that in order to produce a fusion protein, the nucleic acid sequences are linked so that the resulting reading frame does not contain an internal stop codon.

Host: The term "host" is used herein to refer to humans or any non-human animals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, non-human primates) or systems (e.g., cells or cell lines). In some embodiments, the host is an organism to which cells or cell colonies expressing CAR and/or TGFβ modulators described herein are to be administered. In some embodiments, administration of the cell colony results in an improved 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, a host cell can be used to produce a modified CAR molecule as described herein by standard recombinant techniques. The skilled person will understand after reading this disclosure that such terms refer not only to specific subject cells, but also to the offspring of such cells. Since certain modifications may occur in progeny due to mutations or environmental influences, such progeny may not actually be completely identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

In some embodiments, host cells refer to human cells. 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 cells and eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, insect cells infected with baculovirus, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, such as hybridomas or quadruple hybridomas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, HEK293T, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, such as a retinal cell expressing a viral gene (eg, a PER.C6 ^TM cell).

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 stimuli such as antigens or vaccines. The immune response may include any body cell involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and/or adaptive immune responses. Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and/or activity of lymphocytes (e.g., B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, and the like. In some embodiments, the immune response refers to the immune response observed after administration of the armored CAR-T cells or unarmored CAR-T cells described herein. In some embodiments, the immune response observed following administration of the armored CAR-T cells described herein is measured by one or more of: increased proliferation of CAR-expressing cells, increased IFNg production by CAR-expressing cells, increased IL-2 production by CAR-expressing cells, increased proliferation of host immune cells, increased IL-2 production by 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 that occur in an artificial environment (eg, in a test tube or reaction vessel), in cell culture, etc., rather than in a multicellular organism.

In vivo: As used herein, the term "in vivo" refers to events occurring within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

Isolated: As used herein, the term "isolated" refers to a substance and/or entity that is (1) separated from at least some of the components with which it was originally associated when produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured without human intervention. An 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 agent 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 it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, 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.); in such embodiments, such carriers or excipients are not included in the calculation of the separation percentage or purity of the substance. As just one example, in some embodiments, a biopolymer such as a polypeptide or polynucleotide that occurs in nature is considered "isolated" when: a) it is not associated with some or all of the components that accompany it in nature by virtue of its origin or source of derivation; b) it is substantially free of other polypeptides or nucleic acids from the same species as the species in which it is produced in nature; c) it is expressed by, or associated with components of, a cell or other expression system other than that of the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from that in which it is produced in nature is considered an "isolated" polypeptide. In some embodiments, a cell may be an "isolated cell" that is separated from molecules and/or cellular components that naturally accompany the cell. Alternatively or additionally, in some embodiments, a cell that has been subjected to one or more purification techniques may be considered an "isolated" cell to the extent that it has been separated from other components with which it is associated: a) in nature; and/or b) with which it was originally produced.

Linker: 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 connect them to each other. As used herein, a "peptide linker" refers to one or more amino acids used to couple two proteins together (e.g., coupling VH and VL domains).

Modulation or modulator: As used herein, the term "modulation" or "modulator" refers to the ability of a component to positively or negatively alter a related function. Exemplary modulation includes changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%. For example, provided herein is a TGFB signaling modulator that is capable of altering or preventing TGFβ receptor signaling. It will be appreciated by those skilled in the art that this can be achieved by binding to a cytokine (i.e., TGFβ) that activates TGFβR signaling, or by binding to its receptor itself (e.g., a TGFβ antibody or fragment thereof, a TGFBR antibody or fragment thereof). Therefore, this term encompasses two molecules that bind to TGFβ and a molecule that binds to TGFβR. In one embodiment, the modulator of the present disclosure can neutralize TGFβ signaling through TGFβRII. "Neutralization" means blocking the normal signaling effect of TGFβ, so that the presence of TGFβ has a neutral effect on TGFβRII signaling. In some embodiments, the TGFβ modulator improves the 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 an oligonucleotide chain or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is an oligonucleotide chain or a compound and/or substance that can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As is clear 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 chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" is RNA or contains RNA; in some embodiments, "nucleic acid" is DNA or contains DNA. In some embodiments, a nucleic acid is one or more natural nucleic acid residues, contains one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is one or more nucleic acid analogs, contains one or more nucleic acid analogs, 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", which are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds, and are considered to be 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 rather than phosphodiester bonds. 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-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated 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) compared to those in natural nucleic acids. In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product such as an RNA or protein. In some embodiments, nucleic acid includes one or more introns. In some embodiments, nucleic acid is prepared by separating from a natural source, synthesizing (in vivo or in vitro) an enzyme polymerized based on a complementary template, breeding in a recombinant cell or system, 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 complementary sequence to a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles) that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, E.W.Martin, Mack Publishing Co., Easton, PA, 15th edition (1975) describes compositions and preparations suitable for delivering one or more therapeutic compositions as drugs. In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally contain injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, dextrose aqueous solutions, glycerol, etc. as vehicles. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

Polypeptide: A "polypeptide", in general, is a string of at least two amino acids connected to each other by peptide bonds. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is connected to other amino acids by at least one peptide bond. It will be understood by those of ordinary skill in the art that polypeptides sometimes include "non-natural" amino acids or other entities, which nonetheless can be optionally incorporated into a polypeptide chain. In some embodiments, the term "polypeptide" is used to refer to a specific functional category of polypeptides, such as antibodies, chimeric antigen receptors, or co-stimulatory domain polypeptides, etc. For each such category, several examples of amino acid sequences of known exemplary polypeptides within the category are provided in this specification and/or known in the art; in some embodiments, one or more such known polypeptides are reference polypeptides of the category. In such embodiments, the term "polypeptide" refers to any member of a category that shows sufficient sequence homology or identity to a related reference polypeptide, and those skilled in the art will understand that the reference polypeptide should be included in the category. In many embodiments, members of representative categories also share significant activity with the reference polypeptide. For example, in some embodiments, the member polypeptides exhibit an overall sequence homology or identity of at least about 30-40% with the reference polypeptide, and are generally greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or include at least one region (i.e., a conserved region, generally including characteristic sequence elements) that exhibits very high sequence identity, generally greater than 90%, even 95%, 96%, 97%, 98% or 99%. Such conserved regions generally encompass at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids.

It should be understood that the antibodies and antigen-binding agents of the present 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., will 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). "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with 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: As used herein, the term "prevention" refers to preventing, avoiding disease manifestation, delaying onset, 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 if a statistically significant reduction 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, the agent is considered to "prevent" the particular 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 preparation containing more than about 90% of a particular agent or entity is generally considered to be a pure preparation. 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.

Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide (e.g., a polypeptide as described herein) 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 recombinant, combinatorial polypeptide library, or a polypeptide prepared, expressed, created, or isolated by any other means involving splicing of selected sequence elements into one another. In some embodiments, one or more such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements and/or combinations thereof are designed by computer. In some embodiments, one or more such selected sequence elements are produced by a combination of multiple (e.g., two or more) known sequence elements that do not naturally occur in the same polypeptide.

Reference: The term "reference" is often used herein to describe a standard or control agent, individual, colony, sample, sequence or value, and is compared with a reagent, individual, colony, sample, sequence or value of interest. In some embodiments, the test and/or determination of a reference agent, individual, colony, sample, sequence or value is substantially carried out simultaneously with the test or determination of the agent, individual, colony, sample, sequence or value of interest. In some embodiments, a reference agent, individual, colony, sample, sequence or value is an empirical reference, optionally implemented in a tangible medium. Typically, as will be appreciated by those skilled in the art, a reference agent, individual, colony, sample, sequence or value is measured or characterized under conditions similar to those conditions for measuring or characterizing an agent, individual, colony, sample, sequence or value of interest.

Single domain antibody: As used herein, the terms "single domain antibody (sdAb)", "variable single domain" or "immunoglobulin single variable domain (ISV)", "single heavy chain variable domain (VH) antibody" refer to a single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. sdAb is a single antigen-binding polypeptide with three complementary determining regions (CDRs). Only sdAb is able to bind to the 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 "VHH". Certain VHHs may also be referred to as nanobodies. Camelized sdAbs are one of 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); Hassanzadeh-Ghassabeh 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. Camelized VHH domains can be humanized according to standard techniques available in the art, and such domains are considered "domain antibodies". As used herein, VH includes camelized VHH domains, and the term VHH can be used to refer to domain antibodies of human or camelid origin that contain only heavy chains. As explained below, some embodiments of various aspects of the invention relate to binding agents that contain a single heavy chain variable domain antibody/immunoglobulin heavy chain single variable domain that can bind to TGFβ antigen in the absence of a light chain.

Subject: As used herein, the term "subject" refers to any mammal, including humans. In certain embodiments of the present invention, the subject is an adult, a teenager or an infant. In some embodiments, the term "individual" or "patient" is used and is intended to be interchangeable with "subject". The present invention also encompasses the administration of pharmaceutical compositions and/or the conduct of intrauterine treatment methods. For example, the subject may be a patient (e.g., a human patient or an animal patient) with cancer (e.g., gastrointestinal origin), symptoms of cancer, wherein at least some cells express TGFβ, or a patient with a tendency to cancer, wherein at least some cells express TGFβ. Unless otherwise indicated, the term "non-human animal" of 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, etc.

Substantially: As used herein, the term "substantially" refers to the qualitative condition of having the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completion or reach or avoid an absolute result. Thus, the term "substantially" is used herein to encompass the potential lack of completeness inherent in many biological and chemical phenomena.

Therapeutic agent: As used herein, the term "therapeutic agent" refers to a biologically active agent (e.g., an antigen binding agent). The term is used herein to refer to a compound, a mixture of compounds, a biomacromolecule, or an extract made from a biological material. In some embodiments, the therapeutic agent may be an anticancer agent or a chemotherapeutic agent. As used herein, the term "anticancer agent" or "chemotherapeutic agent" refers to an agent with functional properties that inhibit the development or progression of human tumors, particularly malignant (cancerous) lesions, such as carcinomas, sarcomas, lymphomas, or leukemias. Inhibition of metastasis or angiogenesis is typically a property of an anticancer agent or a chemotherapeutic agent. A chemotherapeutic agent may be a cytotoxic agent or a cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation.

Transformation: 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 can rely on any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, a specific transformation method is selected based on the host cell being transformed, and may include, but is not limited to: viral infection, electroporation, mating, lipid transfection. In some embodiments, "transformed" cells are stably transformed because the inserted DNA can replicate as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cells temporarily express the introduced nucleic acid for a limited period of time.

Transforming Growth Factor-β (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 native amino acid sequence of any TGF-β from humans, including latent forms thereof and bound or unbound complexes of precursors and mature TGF-β ("latent TGF-β"). References herein to such TGF-β will be understood as references to any of the currently identified forms, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 and TGF-β5 and their latent forms, as well as human TGF-β species to be identified in the future, including polypeptides derived from the sequence of any known TGF-β and at least about 75%, preferably at least about 80%, more preferably at least about 85%, yet more preferably at least about 90%, and even more preferably at least about 95% homologous to said sequence. The specific terms "TGF-β1", "TGF-β2" and "TGF-β3", as well as "TGF-β4" and "TGF-β5" refer to TGF-β as defined in the literature, such as Derynck et al., Nature, supra; Seyedin et al., J. Biol. Chem., 262, supra; and deMartin et al., supra. The term "TGF-β" refers to the gene encoding human TGF-β. The preferred TGF-β is the human TGF-β native sequence.

Members of the TGF-β family are defined as those that carry nine cysteine residues in the mature portion of the molecule, share at least 65% homology with other known TGF-β sequences in the mature region, and compete for the same receptor. Furthermore, they all appear to be encoded as larger precursors that share a region of high homology near the N-terminus and appear to retain three cysteine residues in the portion of the precursor that is later removed by processing. Furthermore, TGF-β appears to have a four or five amino acid processing site.

Transforming Growth Factor-β 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). The TGFβ receptor is characterized by serine/threonine kinase activity and exists in several different homo- or hetero-dimeric isoforms.

TGFβ signaling pathway regulator or TGFβ regulator: As used herein, the terms "TGFβ signaling pathway regulator" or "TGFβ regulator" used interchangeably herein refer to molecules (e.g., antibodies or fragments thereof) that can regulate the TGFβ signaling pathway (e.g., having an inhibitory, blocking or neutralizing effect), and the molecule can bind to TGFβ itself or it can bind to TGFβ receptors on cells. In either case, the regulator inhibits the TGFβ signaling pathway (e.g., by binding to the cytokine (ie, TGFβ) itself or by binding to a receptor of TGFβ). Therefore, this term encompasses two types of regulators that bind to TGFβ and bind to TGFβ receptors. In various embodiments described herein, TGFβ signaling pathway regulators are expressed in modified immune cells (e.g., CAR-T cells) together with chimeric antigen receptors. CAR-T cells expressing such TGFβ signaling pathway regulators are referred to herein as TGFβ armored CAR-T cells.

Treatment: As used herein, the term "treat" or "treatment" is defined as administering a therapeutic agent to a subject, such as a patient, or administering (e.g., by applying) to a tissue or cell isolated from a subject and returned to the subject. 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). Treatment may be curing, healing, alleviating, mitigating, altering, remedying, improving, alleviating, improving, or affecting the tendency of a disorder, a symptom of a disorder, or a disorder (e.g., cancer). Although 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 disorders, such as disorders described herein (e.g., cancer).

The invention described herein is used for therapeutic, prophylactic or preventive treatment in an "effective amount". A therapeutically effective amount of armored CAR-T cells (e.g., engineered cells co-expressing CAR and TGFβ signaling regulators) described herein is an effective amount that can improve or reduce one or more disease symptoms or 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 an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site. Heavy chain-only antibodies have a single heavy chain variable region.

Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transmitting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which an additional DNA segment can be joined. Another type of vector is a viral vector, in which an additional DNA segment can be joined to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of a gene operably connected thereto. Such vectors are referred to herein as "expression vectors".

DETAILED DESCRIPTION

The present invention provides methods and compositions for enhancing the immune response to cancer using modified immune cells (e.g., CAR-T cells) armored with polypeptides that modulate TGFβ signaling. The present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not used for limitation, unless otherwise specified, because the scope of the present invention will be limited only by the appended claims.

TGF-B/SMAD signaling

Transforming growth factor β (TGF-β) is a multifunctional cytokine, originally named for its ability to transform normal fibroblasts into cells capable of anchorage-independent growth. TGF-β signaling controls many key 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. Dysregulation of TGF-β leads to pathological conditions, such as birth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

TGF-β is mainly produced by hematopoietic and tumor cells, which can regulate (i.e. stimulate or inhibit) the growth and differentiation of cells from a variety of normal and tumor tissue sources (Spom et al., Science, 233: 532 (1986)), and stimulate the formation and development of various matrix elements. TGF-β is involved in many proliferative and non-proliferative cell processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses.

TGF-β also possesses immunosuppressive activities, including lymphokine activated killer cells (LAK), cytotoxic T lymphocyte (CTL) inhibition, reduction of B cell lymphopoiesis and kappa light chain expression, negative regulation of hematopoiesis, downregulation of HLA-DR expression on tumor cells, and inhibition of proliferation of antigen activated B lymphocytes in response to B cell growth factor. Many human tumors and many tumor cell lines produce TGF-β, suggesting a possible mechanism that allows those tumors to evade normal immune surveillance. Together with the observation that some transformed cell lines have lost the ability to respond to TGF-β in an autocrine manner, which stimulates matrix formation and reduces tumor immune surveillance, this negative immunoregulatory effect suggests an attractive model for neoplastic dysregulation and proliferation.

Since TGF-β signaling is important for both healthy cells and cancer regulation, systemic targeting of TGF-β may cause unwanted side effects. Specifically with regard to cancer, members of the TGF-β family are known to have many biological activities associated with tumorigenesis (including angiogenesis) and metastasis. TGF-β inhibits the proliferation of many cell types, including capillary endothelial cells and smooth muscle cells. TGF-β downregulates integrin expression (α1β1, α2β1, and αvβ3, which are involved in endothelial cell migration). Integrins are involved in the migration of all cells, including metastatic cells. TGF-β downregulates the expression of matrix metalloproteinases required for angiogenesis and metastasis. TGF-β induces plasminogen activator inhibitor, which inhibits the protease cascade required for angiogenesis and metastasis. TGF-β induces normal cells to inhibit transformed cells. See, e.g., Yingling et al., Nature Reviews, 3(12):1011-1022 (2004), which discloses that dysregulation of TGF-β is involved in the pathogenesis of multiple diseases (including cancer and fibrosis), and proposes a theoretical basis for evaluating TGF-β signaling inhibitors as cancer therapeutics, biomarkers/diagnostics, small molecule inhibitor structures under development, and targeted drug development models to be applied to their development.

As used herein, the term "TGF-β signaling pathway" is used to describe downstream signaling events attributed to TGF-β and TGF-β-like ligands. For example, in one signaling pathway, the TGF-β ligand binds to and activates the type II TGF-β receptor. The type II TGF-β receptor recruits and forms heterodimers with the type I TGF-β receptor. The resulting heterodimer allows phosphorylation of the type I receptor, 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 the control of the expression of mediators involved in cell growth, cell differentiation, tumor formation, apoptosis, and cell homeostasis. It is also contemplated that other TGF-β signaling pathways can be operated according to the methods described herein.

TGF-β signaling pathway modulators

The present invention provides an immunomodulatory system comprising a TGF-β signaling regulator (e.g., a polypeptide that regulates TGF-β signaling or a nucleic acid sequence that encodes a polypeptide that regulates TGF-β signaling). In some embodiments, the TGF-β signaling regulator causes a cellular response when it binds to TGF-β or a TGF-β receptor. In some embodiments, the TGF-β signaling regulator is secreted from a cell.

In multiple embodiments, the present invention provides modified immune cells (eg, T cells) that express chimeric antigen receptors and TGF-β signaling regulators together. Such regulators may bind to TGF-β itself or to TGF-β receptors. CAR-T cells expressing such regulators are referred to herein as TGF-β armored CAR-T cells.

Anti-TGFβ and anti-TGFβR2 antigen binding molecules

In some embodiments, the TGF-β signaling regulator is an antigen binding molecule (e.g., an antibody or an antigen binding fragment thereof). In some embodiments, the antigen binding molecule (e.g., an antibody or an antigen binding fragment thereof) specifically binds to TGF-β. In some embodiments, the antigen binding molecule (e.g., an antibody or an antigen binding fragment thereof) specifically binds to a TGF-β receptor (TGFβR) (e.g., TGFβR1, TGFβR2).

TGF-β signaling regulators (e.g., anti-TGFβ antibody molecules or anti-TGFβR antibody molecules) may include all or antigen binding subsets of the CDRs or heavy chains 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, US20190119387; and International Patent Application Nos. WO2012093125A1, WO2011/012609, and WO 2017/141208 Al; the entire text of each of which is hereby incorporated by reference. Antigen binding agents useful in the immunomodulatory system described herein include, but are not limited to, antibodies that specifically bind to an antigen (e.g., a 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, bivalent antibodies, trivalent antibodies, etc.

In some embodiments, the immunomodulatory system comprises a TGF-β 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 a sequence provided in Table 1. In some embodiments, the immunomodulatory 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, the TGF-b signaling modulator of the present 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 the TGF-β signaling modulator is a single domain antibody (VH).

In some embodiments, the TGF-β signaling regulator comprises a leader sequence. In some embodiments, the TGF-β signaling regulator is a monomer. In some embodiments, the TGF-β signaling regulator is a dimer. In some embodiments, the TGF-β signaling regulator is a trimer.

In some embodiments, the TGF-β signaling regulator comprises a linker that connects domains in tandem. 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 regulator 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 the VL sequence provided in Table 1. In some embodiments, the anti-TGFβ antigen binding agent of the invention comprises a heavy chain variable region amino acid sequence that is identical to the VH sequence provided in Table 1.

In some embodiments, the TGF-β signaling modulators of the invention comprise 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 the vH sequence provided in Table 1. In some embodiments, the TGF-β signaling modulators of the invention comprise a heavy chain variable region amino acid sequence that is identical to 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-TGFβ and anti-TGFβR2 antigen binding molecules

In some embodiments, the anti-TGFβ or anti-TGFβR antigen binding agent is an antibody. The typical structural unit of naturally occurring mammalian antibodies is a tetramer. Each tetramer consists of two pairs of polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains can be divided into κ and λ light chains. Heavy chains can be divided into μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected 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 Fundamental Immunology Chapter 7 (Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989)). The variable region of each light/heavy chain pair forms the antibody binding site. The preferred isotype of the anti-TGFβ antibody molecule is the IgG immunoglobulin, which can be divided into four subtypes, IgG1, IgG2, IgG3 and IgG4, each with a different gamma heavy chain. Most therapeutic antibodies are human, chimeric or humanized antibodies of the IgG1 type. In a specific 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, a complete IgG antibody has two identical binding sites. However, bifunctional or bispecific antibodies are artificial hybrid constructs that have 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 (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from each pair of two chains are aligned by the framework regions to enable binding to a specific epitope. From N-terminus to C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acids of each domain are designated according to the definitions 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 (HCDR1, HCDR2, HCDR3) and the light chain (LCDR1, LCDR2, LCDR3).

Thus, in one embodiment, the antibody molecule comprises one or both of the following:

(a) one, two, three or antigen binding number of the light chain CDRs (LCDR1, LCDR2 and/or LCDR3) of one of the above human hybridomas, selected lymphocytes or murine antibodies. 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 in which one to seven amino acids are conservatively substituted; LCDR2 or modified LCDR2 in which one or two amino acids are conservatively substituted; or LCDR3 or modified LCDR3 in which one or two amino acids are conservatively substituted; and

(b) one, two, three or antigen-binding number of heavy chain CDRs (HCDR1, HCDR2 and/or HCDR3) of one of the above human hybridomas, selected lymphocytes or murine antibodies. In an embodiment, the CDR may comprise one or more or all of the following HCDR1-3 amino acid sequences: HCDR1 or modified HCDR1, wherein one or two amino acids are conservatively substituted; HCDR2 or modified HCDR2, wherein one to four amino acids are conservatively substituted; or HCDR3 or modified HCDR3, wherein one or two amino acids are conservatively substituted.

In some embodiments, the anti-TGFβ antibody molecules or anti-TGFβR (e.g., anti-TGFβR2) antibody molecules of the present invention can cause cells expressing TGFβ (e.g., tumor cells) to produce antibody-dependent cellular cytotoxicity (ADCC). Due to their ability to bind to Fc receptors, antibodies with IgG1 and IgG3 isotypes can be used to induce effector functions of antibody-dependent cellular cytotoxicity. Antibodies with IgG2 and IgG4 isotypes can be used to minimize ADCC reactions because of their low ability to bind to Fc receptors. In a related embodiment, substitutions or changes in glycosylation composition can be made in the Fc region of the antibody, for example, by growing in a modified eukaryotic cell line to enhance Fc receptor recognition, binding and/or cytotoxicity of cells to which the anti-TGFβ antibody or anti-TGFβR (e.g., anti-TGFβR2) antibody binds (see, e.g., U.S. Pat. Nos. 7,317,091, 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, antibodies or antigen-binding fragments (e.g., humanized antibodies, human antibodies) may include amino acid substitutions or replacements that alter or tailor functions (e.g., effector functions). For example, a constant region of human origin (e.g., a γ1 constant region, a γ2 constant region) may 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.), all of which are incorporated herein by reference in their entirety). Preferably, the amino acid sequence of the constant region of human origin containing such amino acid substitutions or replacements is at least about 95% identical to the amino acid sequence of the unaltered constant region of human origin over the entire length, more preferably, at least about 99% identical to the amino acid sequence of the unaltered constant region of human origin over the entire length. Additional anti-TGFβ antigen binding molecules are further described in U.S. Pat. No. 8,785,600 (Nam et al.), all of which are incorporated herein by reference.

In another embodiment, effector function can also be changed by adjusting the glycosylation pattern of the antibody. Change refers to deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that do not exist in the antibody. For example, antibodies with enhanced ADCC activity and mature carbohydrate structures are described in U.S. Patent Application Publication No. 2003/0157108 (Presta), and the structure lacks fucose connected to the Fc region of the antibody. Also referring to U.S. Patent Application Publication No. 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Glycofi has also developed a yeast cell line that can produce antibody-specific glycoforms.

Additionally or alternatively, antibodies with altered glycosylation types may be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications may be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and may be used as host cells, which are engineered to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 (Hang et al.) describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication No. WO03/035835 (Presta) describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in antibodies expressed in the host cells being hypofucosylated (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication No. WO 99/54342 (Umana et al.) describes a cell line engineered to express a glycoprotein modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Humanized antibodies can also be prepared using CDR transplantation methods. The technology for producing such humanized antibodies is known in the art. Generally, humanized antibodies are produced by obtaining nucleic acid sequences encoding variable heavy and variable light chain sequences of antibodies that bind to TGFβ, identifying complementary determining regions or "CDRs" in the variable heavy and variable light chain sequences, and transplanting the CDR nucleic acids to human framework nucleic acid sequences. (See, for example, U.S. Patents Nos. 4,816,567 and 5,225,539). The positions of CDR and framework residues can be determined (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department 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 immunomodulatory system of the present invention comprises a nucleic acid sequence of an anti-TGFβ or anti-TGFβR antibody molecule encoding a CDR from an antibody molecule described in Table 1. In some embodiments, the sequence from Table 1 can be incorporated into a molecule that recognizes TGFβ or TGFβR for use in the treatment methods described herein (e.g., an immunomodulatory system, an immunoresponsive cell, or a treatment method comprising the same). The selected human framework is a framework suitable for in vivo administration, which means that it does not exhibit immunogenicity. For example, such a determination can be made through previous experience of in vivo use of such antibodies and amino acid similarity studies. Suitable framework regions can be selected from human-derived antibodies that have at least about 65% 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), and preferably at least about 70%, 80%, 90% or 95% amino acid sequence identity. A suitable amino acid sequence alignment algorithm, such as CLUSTAL W, can be used to determine amino acid sequence identity using preset parameters. (Thompson J.D. et al., Nucleic Acids Res. 22:4673-4680 (1994)).

Once the CDR and FR of the cloned antibody to be humanized are identified, the amino acid sequence encoding the CDR can be identified, and the corresponding nucleic acid sequence can be transplanted to the selected human FR. This can be done using known primers and linkers, the selection of which is known in the art. All CDRs of a particular human antibody can be replaced by at least a portion of a non-human CDR, or only some CDRs can be replaced by non-human CDRs. It is only necessary to replace the number of CDRs required for the humanized antibody to bind to a predetermined antigen. After the CDRs are transplanted to the selected human FRs, the resulting "humanized" variable heavy chain and variable light chain sequences are expressed to produce humanized Fv or humanized antibodies that bind to TGFβ or TGFβR. Preferably, the CDR-transplanted (e.g., humanized) antibody binds to TGFβ or TGFβR with an affinity similar to, substantially the same or better than that of the donor antibody. Typically, the humanized variable heavy and light chain sequences are expressed as fusion proteins with human constant domain sequences, thereby obtaining complete antibodies that bind to TGFβ. However, humanized Fv antibodies can be generated that do not contain such constant sequences.

Humanized antibodies in which specific amino acids have been replaced, deleted or added are also within the scope of the present invention. In particular, humanized antibodies may have amino acid substitutions in the framework region, for example to improve binding to the antigen. For example, a selected, small number of acceptor framework residues of humanized immunoglobulin chains may be replaced by corresponding donor amino acids. The replacement position includes amino acid residues adjacent to the CDR, or amino acid residues that can interact with the CDR (see, for example, U.S. Patent Nos. 5,585,089 or 5,859,205). The acceptor framework may be a mature human antibody framework sequence or a consensus sequence. As used herein, the term "consensus sequence" refers to the most common or most common sequence designed by the most common residues at each position of the sequence of a region in a related family member. There are a variety of human antibody consensus sequences available, including consensus sequences of different subgroups of human variable regions (see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)). The Kabat database and its applications are freely available online, for example, via IgBLAST at the National Center for Biotechnology Information, Bethesda, Md. (see also Johnson, G. and Wu, T.T., Nucleic Acids Research 29:205-206 (2001)).

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, any of such antibodies can be easily isotype-switched to produce a human IgG4 isotype, for example, while still having the same variable regions (which define the specificity and affinity of the antibody, to some extent). Therefore, when antibody candidates are generated that meet the desired "structural" attributes as discussed above, they can often provide at least some of the additional "functional" attributes desired via isotype switching.

scFv

Single-chain antibodies lack some constant domains or all constant domains of the complete antibody from which they are derived. Therefore, they can overcome some problems associated with the use of complete antibodies. For example, single-chain antibodies tend not to contain some unwanted interactions between the heavy chain constant region and other biomolecules. In addition, single-chain antibodies are significantly smaller than complete antibodies and can have greater permeability compared to complete antibodies, so that single-chain antibodies are more effectively positioned and incorporated into the target antigen binding site. In addition, compared to complete antibodies, the relatively small size of single-chain antibodies makes it unlikely to cause an inappropriate immune response in recipients.

In some embodiments, the TGFβ signaling regulator is a single-chain antigen binding molecule (e.g., scFv) that specifically binds to TGFβ. In some embodiments, the TGFβ signaling regulator is a single-chain antigen binding molecule (e.g., scFv) that specifically binds to a TGF-B receptor (TGFβR) (e.g., TGFβR1, TGFβR2).

A plurality of single-chain antibodies (each having a VH and a VL domain covalently linked by a first peptide linker) can be covalently linked by at least one or more peptide linkers to form a multivalent single-chain antibody, and it can be monospecific or multispecific. Each chain of the multivalent single-chain antibody includes a variable light chain fragment and a variable heavy chain fragment, and is linked to at least another 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 can be combined to form a bivalent antibody also referred to as a bivalent dimer. A bivalent antibody has two chains and two binding sites and can have monospecificity or bispecificity. Each chain of the bivalent antibody includes a VH domain connected to a VL domain. The domains are connected to sufficiently short linkers to prevent pairing between domains on the same chain, thereby promoting pairing between complementary domains on different chains, thereby reproducing two antigen binding sites.

Three single-chain antibodies can be combined to form a trivalent antibody, also known as a trivalent trimer. A trivalent antibody is composed of the amino acid terminus of a VL or VH domain fused directly to the carboxyl terminus of a VL or VH domain (i.e., without any linker sequence). A trivalent antibody has three Fv heads, in which the polypeptides are arranged in a circular, head-to-tail manner. A possible trivalent antibody conformation is planar, in which the three binding sites are located in a plane at an angle of 120 degrees to each other. A trivalent antibody can have monospecificity, bispecificity, or trispecificity.

Single domain antibodies

Single domain antibodies (sdAbs) differ from conventional 4-chain antibodies in that they have a single monomeric antibody variable domain. For example, camelids and sharks produce sdAbs called heavy chain-only antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of a camelized heavy chain-only antibody has a single heavy chain variable domain (VHH), which can have a high affinity for antigen without the help of a light chain. Camelized VHHs are known as the smallest functional antigen-binding fragments, with a molecular weight of approximately 15 kD.

One aspect of the present application provides an isolated single domain antibody (referred to herein as "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 anti-TGFβR sdAb described herein, and antigen-binding proteins comprising any anti-TGFβR sdAb 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, can be used in another antigen binding agent, such as in a CDR graft, a humanized or chimeric antibody molecule. Embodiments include antibody molecules comprising enough CDRs (e.g., all three CDRs from one of the above-mentioned heavy chain variable regions) to bind to TGFβ.

In some embodiments, the CDRs (e.g., all HCDRs) are embedded in human or human-derived framework regions. Examples of human framework regions include human germline framework sequences, human germline sequences that have been affinity matured (in vivo or in vitro), or synthetic human sequences, such as consensus sequences. In one embodiment, the heavy chain framework is an IgG1 or IgG2 framework.

In some embodiments, the TGFβ modulators of the 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 (eg, an antigen binding agent that does not comprise an immunoglobulin light chain).

Antibody fragments for in vivo therapeutic or diagnostic purposes 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 include one, two or more linear or branched moieties selected from: hydrophilic polymer groups (e.g., linear or branched polymers (e.g., polyalkylene glycols such as polyethylene glycol, monomethoxy-polyethylene glycol, etc.), carbohydrates (e.g., dextran, cellulose, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyaspartic acid, etc.), polyalkane oxides and polyvinylpyrrolidone), fatty acid groups (e.g., monocarboxylic acids or dicarboxylic acids), fatty acid ester groups, lipid groups (e.g., diacylglycerol groups, sphingolipid groups (e.g., ceramide groups)) or phospholipid groups (e.g., phosphatidylethanolamine groups). Preferably, the organic moiety is bound 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 500 Da to about 50,000 Da, 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 in, for example, U.S. Pat. Nos. 4,179,337 and 5,612,460, PCT Publications Nos. WO 95/06058 and WO 00/26256, and U.S. Patent Application Publication No. 20030026805.

TGFβR extracellular domain

It is contemplated that the TGF-β receptor used in the immunomodulatory system described herein may be any TGF-β receptor, including receptors from the activin-like kinase family (ALK), the bone morphogenetic protein (BMP) family, the Nodal family, the growth and differentiation factor family (GDF), and the TGF-β receptor family. The TGF-β receptor is a serine/threonine kinase receptor that affects various growth and differentiation pathways in cells. In some embodiments, the TGFβ signaling regulator is an engineered recombinant extracellular domain (ECD) of a TGFβ receptor (e.g., TGFβR1, TGFβR2). In some embodiments, the TGF-β receptor that can be used in the immunomodulatory system described herein is a type II TGF-β receptor (e.g., TGF-βR2).

In some embodiments, the TGFβ modulator comprises a TGFβR provided in Table 2. In some embodiments, the TGFβ modulator comprises a sequence that is 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 TGFβR extracellular domains

Chimeric Antigen Receptor

In some aspects, the present invention provides an immunomodulatory system comprising a TGF-β signaling regulator (e.g., a polypeptide that regulates TGF-β signaling or a nucleic acid sequence that encodes a polypeptide that regulates 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) an extracellular antigen binding motif, (2) a connection/transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso W M, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2(4): e23621). In some embodiments, the CAR of the present invention comprises a signal or leader peptide, an antigen binding domain, a transmembrane and/or hinge domain, a co-stimulatory domain, and an intracellular domain from the N-terminus to the C-terminus. In some embodiments, CAR is a "first generation CAR", for example, including a CAR that only provides a CD3ζ signal when an antigen is bound. "Second generation CAR" includes CARs that provide co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ) simultaneously. "Third generation CAR" includes CARs that provide multiple co-stimulation (e.g., CD28 and CD137) and activation (CD3). In multiple embodiments, CAR is selected to have high affinity or avidity for antigens.

The antigen binding motif of CAR is usually formed after the minimum binding domain or single domain antibody of single-chain fragment variable domain (ScFv), immunoglobulin (Ig) molecule (for example, WO2018/028647A1). Alternative antigen binding motifs, such as receptor ligands (that is, IL-13 has been engineered to bind to tumor-expressing IL-13 receptors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (such as NKG2D) are also engineered. Alternative cell targets (such as NK or γ-δ T cells) for CAR expression are also under development (Brown CE et al., Clin Cancer Res.2012; 18 (8): 2199-209; Lehner M et al., PLoS One.2012; 7 (2): e31210).

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 CD3 ζ intracellular domain from N-terminus to C-terminus.

The connecting motif of CAR can be a relatively stable domain, such as the constant domain of IgG, or a flexible linker designed to be extended. Structural motifs, such as those derived from IgG constant domains, can be used to extend the ScFv binding domain away from the T cell membrane surface. This may be important for certain tumor targets whose binding domains are particularly close to the surface membrane of tumor cells (e.g., disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motifs used in CARs have always included the CD3-ζ chain because 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 containing a CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009; 183 (9): 5563-74). With the emergence of new technologies, T cells are activated with beads linked to anti-CD3 and anti-CD28 antibodies, and the emergence of the classic "signal 2" from CD28 no longer needs to be encoded by CAR itself. Using bead activation, it was found that the third-generation vectors were not superior to the second-generation vectors in in vitro assays, and they were not significantly superior to the second-generation vectors in leukemia mouse models (Haso W, Lee D W, Shah NN, 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 CD19-specific CAR in the second generation CD28/CD3-ζ (Lee D W et al., American Society of Hematology Annual Meeting. New Orleans, La.; Dec. 7-10, 2013) and CD137/CD3-ζ signaling format (Porter D L et al., N Engl J Med. 2011; 365 (8): 725-33) proves this point. In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 can also provide important continuous signals in CAR-transduced T cells (Yvon E et al., Clin Cancer Res. 2009; 15 (18): 5852-60). Equally important is the culture conditions for culturing CAR T cell populations.

The characteristics of CAR include its ability to redirect T cell specificity and reactivity to selected targets in a non-MHC restricted manner and to exploit the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition enables T cells expressing CAR to recognize antigens that are not related to antigen processing, thus bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR will advantageously not dimerize the exogenous T cell receptor (TCR) α and β chains.

Extracellular domain

As described herein, the CAR comprises a target-specific binding element, which is also referred to as an antigen binding domain or part. The selection of the domain depends on the type and number of the ligands defining the surface of the target cell. For example, the antigen binding domain can be selected to identify ligands (eg, cancer antigens) as cell surface markers on target cells associated with a specific disease state (eg, cancer). Therefore, examples of cell surface markers that can be used as ligands of antigen binding domains in CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

In some embodiments, the extracellular domain of CAR comprises an antigen binding agent that specifically binds to a cancer antigen. In certain embodiments, CAR binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Antigen sources include, but are not limited to, cancer proteins. Antigens can be expressed as peptides or complete proteins or portions thereof. Complete proteins or portions thereof can be natural or mutated. Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, 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 antigens), 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 (GD2), ganglioside G3 (GD3), guanylate cyclase (Guanylyl cyclase) C (GCC), human epidermal growth factor receptor 2 (ITER-2), human telomerase reverse transcriptase (hTERT), interleukin 13 receptor subunit alpha-2 (IL-13Rcx2), k-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survival protein, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, tumor embryonic antigen (h5T4), 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-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

In certain embodiments, the CAR is bound to a CD19 polypeptide. In certain embodiments, the CAR is bound to a human CD19 polypeptide. In certain embodiments, the CAR is bound to the extracellular domain of a CD19 protein. In certain embodiments, the CD19 CAR comprises a sequence 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 the extracellular domain of a GCC protein. In certain embodiments, the anti-GCC CAR comprises the sequence 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 the extracellular domain of a mesothelin protein.

In certain embodiments, the CAR is bound to a pathogen antigen, for example, to treat and/or prevent, for example, a pathogen infection or other infectious disease in an immunocompromised subject. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa capable of causing disease.

Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HDTV-III, LAVE or HTLV-IIELAV, or HIV-III; and other isolates, such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, German measles virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Baculoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hanjiang virus, bunga ) virus, phlebovirus, and Nairavirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Diribonuclear Viridae; Hepaciviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papiopolyomaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (simple herpesviruses); Herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes simplex virus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., the causative agent of hepatitis delta (thought to be a deleted satellite of hepatitis B virus), the causative agent of non-A, non-B hepatitis (category 1 = transmitted internally; category 2 = transmitted parenterally (i.e., hepatitis C); Norwalk and related viruses, and astroviruses).

The non-limiting examples of bacteria include Pasteurella, Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas and Salmonella. The specific examples of infectious bacteria include but are not limited to Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria meningitidis, and so on. tidis), Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus mon iliformis, Treponema palladium, Treponema tenuissima, Leptospira, Rickettsia, and Actinomyces israelii.

In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in 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 joint. In some embodiments, the joint comprises GGGGS (SEQ ID NO: 59). In some embodiments, the 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 joint 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; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody (sdAb), a camelized antibody; a masked antibody, a small modular immunopharmaceutical ("SMIPsTM"), a single chain, a tandem bivalent antibody, a VHH, an anticalin, a nanobody, a humanized antibody, a minibody, a BiTE, an ankyrin repeat protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an Adnectin, an Affilin, a penetrating antibody; an affinity antibody, a TrimerX, a miniprotein, a Fynomer, a Centyrin; and a 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. Regarding 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 a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein.

The transmembrane region used in the CAR described herein may be derived from (i.e., comprising at least the following transmembrane region) α, β or ζ chain of the T cell receptor, CD28, CD3ε, 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 mainly contain 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, may form a bond between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In some embodiments, the linker is a glycine-serine doublet or an alanine triplet linker.

In some embodiments, in addition to the transmembrane domain as described above, a transmembrane domain naturally associated with one of the domains of CAR is used. In some embodiments, the transmembrane domain can be selected by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

In some embodiments, the transmembrane domain in the CAR of the present invention is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 42. In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence SEQ ID NO: 42. In some embodiments, the sequence included in the transmembrane domain has at least one, two or three modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO: 42 but no more than 20, 10 or 5 modifications (e.g., substitutions), or its sequence is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 42.

In CAR, the spacer domain (also referred to as hinge domain) can be configured between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide for connecting the transmembrane domain to the extracellular domain and/or the transmembrane domain to the 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 can 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 is increased. Exemplary spacers are known to those of ordinary skill in the art and include those described in U.S. Pat. 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, No. 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 those listed in 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 the binding of CAR to antigens and improves signal transduction to cells. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can 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 a nucleic acid sequence IEVMYPPPYLDNEKSNGTIIHV KGKHLCPSPLFPGPSKP (SEQ ID NO: 41). In some embodiments, the hinge domain comprises a nucleic acid sequence encoding an amino acid sequence SEQ ID NO: 41. In some embodiments, the sequence included in the hinge domain has at least one, two or three modifications (eg, substitutions) in the amino acid sequence of SEQ ID NO: 41 but no more than 20, 10 or 5 modifications (eg, substitutions), or its sequence is 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 domain are derived from different molecules (e.g., CD8 is fused to CD28). In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a nucleic acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV GGVLACYSLLVTVAFIIFWV (SEQ ID NO: 43). In some embodiments, the hinge domain comprises a nucleic acid sequence encoding an amino acid sequence SEQ ID NO: 43. In some embodiments, the sequence included in the hinge domain has at least one, two or three modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO: 43 but no more than 20, 10 or 5 modifications (e.g., substitutions).

Intracellular domain

The cytoplasmic domain or intracellular signaling domain of CAR is responsible for activating at least one normal effector function of the immune cell in which CAR is placed. The term "effector function" refers to a special function of a cell. For example, the effector function of a T cell may be cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a part of a protein that transduces effector function signals and guides cells to perform specific functions. Although a complete intracellular signaling domain can usually be used, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the complete chain as long as it can transduce the effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce an effector function signal.

Examples of intracellular signaling domains for CAR include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which work together to initiate signal transduction after antigen receptor engagement, and any derivatives or variants of these sequences, and any synthetic sequences with the same function. The signal generated by TCR alone is not enough to fully activate T cells, and secondary or costimulatory signals are also required. Therefore, T cell activation can be referred to as being mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence acting in a stimulatory manner may include a signaling motif, which is referred to as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs disclosed herein containing primary cytoplasmic signaling sequences particularly for CAR include those derived from TCR ζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAMs include amino acid numbers 51 to 164 of CD3ζ (NCBI RefSeq: NP.sub.--932170.1), amino acid numbers 45 to 86 of Fcε.RI.γ (NCBI RefSeq: NP.sub.--004097.1), amino acid numbers 201 to 244 of Fcε.RI.β (NCBI RefSeq: NP.sub.--000130.1), amino acid numbers 139 to 182 of CD3γ (NCBI RefSeq: NP.sub.--000064.1), amino acid numbers 128 to 171 of CD3δ (NCBI RefSeq: NP.sub.--000723.1), amino acid numbers 153 to 207 of CD3ε (NCBI RefSeq: NP.sub.--000724.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--000136.1). RefSeq: NP.sub.--055022.2), amino acid numbers 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acid numbers 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acid numbers 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and amino acid numbers 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), and variants thereof having the same function as these peptides. The amino acid numbering based on the amino acid sequence signal of the NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). 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 CAR can be designed to include the CD3-ζ signaling domain itself, or in combination with any other desired cytoplasmic domain available in the context of CAR. For example, the intracellular domain of CAR may include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a CAR that includes an 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 effectively respond to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc. Specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acid numbers 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acid numbers 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acid numbers 207 to 235 of CD8α (NCBI RefSeq: NP.sub.--001759.3), amino acid numbers 196 to 210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--001759.3), amino acid numbers 196 to 210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--001759.2 ... RefSeq: NP.sub.-006130.1), amino acid numbering 214 to 255 of CD137 (4-1BB, NCBIRefSeq: NP.sub.--001552.2), amino acid numbering 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1) and amino acid numbering 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides. Therefore, although the disclosure herein mainly uses 4-1BB as an example of a costimulatory signaling element, other costimulatory elements are also within the scope of the present disclosure.

The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a linkage. In some embodiments, the linker is a glycine-serine doublet or an alanine triplet linker.

In some embodiments, the intracellular domain is designed to include a CD28 co-stimulatory signaling domain. In some embodiments, the intracellular domain of the CAR includes 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 include the signaling domain of CD3-ζ and the signaling domain of CD28.

In some embodiments, the intracellular domain includes CD3-ζ with one or more modified immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the intracellular domain includes CD3-ζ, which has three immunoreceptor tyrosine-based activation motifs (ITAMs), wherein the first is unmodified, and the second and third ITAMs are changed and named "1XX." In some embodiments, the amino acid sequence included in the intracellular domain of the CAR is RVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIG MKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 45) at least 95%, 96%, 97%, 98%, 99% or 100% identical.

In some embodiments, the intracellular signaling domain comprised by the CAR comprises a modified CD3z polypeptide (e.g., a modified human CD3z polypeptide), wherein the modified CD3z polypeptide comprises native ITAM1, native BRS1, native BRS2, native BRS3, an ITAM2 variant having two loss-of-function mutations, and an ITAM3 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide).

In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domains of CD28 and 4-1BB.

In some embodiments, the intracellular domain of the CAR is designed to contain 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 the amino acid sequence provided in Table 3.

Table 3. Exemplary chimeric antigen receptors

Functional characteristics of CAR

The functional part of CAR disclosed herein is also explicitly included in the scope of the present invention. The term "functional part" when used to refer to CAR refers to any part or fragment of one or more CAR disclosed herein, and the part or fragment retains the biological activity of the CAR (parent CAR) of the part. The functional part covers, for example, those parts of CAR, which are similar to, the same degree or a higher degree of ability to recognize target cells or detect, treat or prevent diseases as the parent CAR. With regard to parent CAR, the functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

The functional portion may include additional amino acids at the amino or carboxyl terminus or both termini of the portion, which are not found in the amino acid sequence of the parent CAR. Ideally, the additional amino acids do not interfere with the biological function of the functional portion, for example, identifying target cells, detecting cancer, treating or preventing cancer, etc. More ideally, the additional amino acids enhance biological activity compared to the biological activity of the parent CAR.

The scope of the present disclosure includes functional variants of CAR disclosed herein. As used herein, the term "functional variant" refers to a CAR, polypeptide or protein having substantially or significantly sequence identity or similarity to a parent CAR, and the functional variant retains the biological activity of the CAR mutated therefrom. Functional variants include those variants of CAR (parent CAR) such as those described herein, which retain the ability to recognize target cells similar to, the same degree or a higher degree of the parent CAR. With regard to parent CAR, the functional variant may be identical to the amino acid sequence of the parent CAR, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more.

Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. Alternatively or additionally, functional variants may comprise the amino acid sequence of a parent CAR having 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 can enhance the biological activity of the functional variant, so 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 with a specific physical and/or chemical property is exchanged for another amino acid with the same or similar chemical or physical properties. For example, the conservative amino acid substitutions may be substitutions of an acidic/negatively charged polar amino acid by another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain by another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid by another basic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain by another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain by another amino acid with a β-branched side chain (e.g., He, Thr and Val), an amino acid with an aromatic side chain by another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

The CAR may 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 parts and functional variants) may have any length, that is, may contain any number of amino acids, provided that the CAR (or its functional part or functional variant) retains its biological activity, for example, 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 may be about 50 to about 5000 amino acids in length, for example, 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 the functional part and functional variant of the present invention) may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example: aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole Indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and a-tert-butylglycine.

The CAR (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized or conjugated.

Substitutions and variants

In some embodiments, amino acid sequence variants of antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. The amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution of residues in the antibody amino acid sequence. Any combination of deletion, insertion and substitution can be performed to complete the final construct, provided that the final construct has desirable features, such as antigen binding.

a) Substitution, insertion and deletion variants

In some embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include HVR and FR. The amino acid side chain categories are further described below. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as antigen binding retention/improvement, immunogenicity reduction, or ADCC or CDC improvement.

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study 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 produced, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activities (e.g., binding affinity).

Changes (e.g., substitutions) may be made in HVRs, for example, to improve 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., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), wherein the binding affinity of the resulting variant VH or VL is tested. Affinity maturation is achieved by construction and rescreening from a secondary library, as described in, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ (2001)). In some embodiments of affinity maturation, diversity is introduced in variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain 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 method for introducing diversity involves an HVR-guided approach, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are typically targeted in particular.

In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within the HVR. Such changes may be outside the HVR "hot spots" or CDRs. 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.

A method for identifying residues or regions in antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or residue group of the target residue (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact sites between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino and/or carboxyl 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 insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for 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 extent to which the antibody is glycosylated.Adding or deleting glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody includes the Fc region, the carbohydrate connected thereto can be changed. The natural antibody produced by mammalian cells generally includes branched, biantennary oligosaccharides, which are generally connected to the Asn297 of the CH2 domain of the Fc region by N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose connected to GlcNAc in the "trunk" of the biantennary oligosaccharide structure. In some embodiments, the antibody of the present application may be oligosaccharide-modified to create antibody variants with certain improved properties.

In some embodiments, antibody variants having carbohydrate structures are provided that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may 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 Asn297 sugar chain, relative to the sum of all sugar structures (e.g., complex, hybrid, and highly mannose structures) attached to Asn 297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence differences in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of disclosures relating to "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; 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. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003/0157108A1, Presta, L; and WO 2004/056312 A1, Adams et al.), and knockout cell lines, such as α-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).

The antibody variant further has a bisected oligosaccharide, for example, wherein the biantennary oligosaccharide connected to the antibody Fc region is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide connected to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

CAR (including its functional parts and functional variants) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for synthesizing polypeptides and proteins from scratch are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R., 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 nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd 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 parts and functional variants thereof) can be isolated and/or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans), etc. Methods of separation and purification are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by a factory. In this regard, CARs can be synthetic, recombinant, isolated and/or purified.

Detectable markers and labels

CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof may also be expressed (e.g., co-expressed) with tag proteins. 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 nucleic acid sequences 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 a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% the same sequence thereof.

CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof may also be conjugated to detectable markers; 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) scanning, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, detectable markers that can be used include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP) can also be used. CAR, T cells expressing CAR, antibodies or their antigen-binding portions can also be conjugated with enzymes such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. for detection. When CAR, T cells expressing CAR, antibodies, and antigen-binding fragments thereof are conjugated with detectable enzymes, they can be detected by adding additional reagents, and the enzyme uses the additional reagents to produce a distinguishable reaction product. For example, when the horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine will produce a colored reaction product that can be visually detected. CAR, T cells expressing CAR, antibodies or their antigen-binding portions can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with an enzyme or a fluorescent marker.

CAR, T cells expressing CAR, antibodies or antigen-binding fragments thereof can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags) that can be recognized by secondary reporter genes.

CAR, T cells expressing CAR, antibodies or antigen-binding fragments thereof may also be conjugated to radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more antigens disclosed herein and cells expressing antigens by x-rays, emission spectroscopy or other diagnostic techniques. In addition, the radiolabels can be used therapeutically as toxins to treat tumors in subjects, such as neuroblastoma. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I.

Methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing an enzyme and a substrate, and detecting the reaction product produced by the action of the enzyme on the substrate, and detecting the chromogenic label by simply observing the colored label.

Immunoreactive cells and host cells

One aspect of the present application provides an engineered immune effector cell (e.g., an immunoreactive cell). As used herein, "immunoreactive cells" refer to cells or progenitor cells or their progeny that play a role in an immune response. In some embodiments, the immunoreactive cells include an immunomodulatory system as 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 regulate TGF-β signaling). In some embodiments, the immunoreactive cells include an immunomodulatory system as described herein (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs); and nucleic acid sequences encoding polypeptides that regulate TGF-β signaling).

In some embodiments, the immune effector cell is a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell or an embryonic stem cell. In some embodiments, the immunoreactive cell is a T cell.

For the purposes of this article, the T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or from a cultured T cell line, such as a T cell of Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or liquids. T cells can also be enriched or purified. The T cell can be a human T cell. The T cell can be a T cell separated from the human race. The T cell can be any type of T cell and can be any developmental stage, including but not limited to: CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+T cells (for example, cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), primitive T cells, etc. The T cell can be a CD8+T cell or a CD4+T cell.

In one embodiment, CAR as described herein can be used in suitable non-T cells. Such cells are those with 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 recombinant expression vector described herein. As used herein, the term "host cell" refers to any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, an animal, a fungus or an algae, or may be a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism such as a human. The host cell may be an attached cell or a suspended cell (i.e., a cell grown in suspension). Suitable host cells are known in the art, and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. 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. While the host cell may be any cell type, may be derived from any type of tissue and may be at any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

One embodiment also provides a cell colony comprising at least one host cell as described herein. The cell colony may be a heterogeneous colony, comprising a host cell containing any of the recombinant expression vectors described, and at least one other cell (e.g., a host cell (e.g., T cell) not comprising any of the recombinant expression vectors) or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell colony may be a substantially homogeneous colony, wherein the colony mainly comprises a host cell containing the recombinant expression vector (e.g., substantially consisting of it). The colony may also be a clonal colony of cells, wherein all cells of the colony are clones of a single host cell comprising a recombinant expression vector, so that all cells of the colony comprise the recombinant expression vector. In one embodiment of the present invention, the cell colony is a clonal colony comprising a host cell, and the host cell comprises a recombinant expression vector as described herein.

CAR (including its functional part and its variant), nucleic acid, recombinant expression vector, host cell (including its population) and antibody (including its antigen binding portion), can be separated and/or purified. For example, a purified (or separated) host cell preparation is one in which the host cell is purer than the cell in its natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell accounts for at least about 50% of the total cell content of the preparation, for example, at least about 70%. 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 Acids and Expression Vectors

One embodiment of the present invention further provides a nucleic acid comprising a nucleotide sequence encoding any CAR, antibody, or antigen binding portion thereof (including functional portions and functional variants thereof) described herein. The nucleic acid of the present invention may include 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 can increase the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing another codon encoding the same amino acid with a natural codon, but can be translated by tRNA that is more readily available in the cell, thereby improving translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

In one embodiment of the invention, the nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, the nucleic acid may include a codon-modified nucleotide sequence encoding any one of the CARs described herein (including its functional parts and functional variants).

Expression vectors include plasmids, retroviruses, cosmids, YACs, EBV-derived episomes, etc. A convenient vector is a vector encoding a functionally complete human CH or CL immunoglobulin sequence with an engineered appropriate restriction site so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site inserted into the J region and the splice acceptor site before the human C region, and also occurs in the splice region within the human CH exon. Suitable expression vectors may include many components, such as an origin of replication, a selectable marker gene, one or more expression control elements such as transcription control elements (e.g., promoters, enhancers, or terminators), and/or one or more translation signals, signal sequences, or leader sequences, etc. Polyadenylation and transcription termination occur at the natural chromosomal site downstream of the coding region. The resulting chimeric antibody can be connected to any strong promoter. Examples of suitable vectors that can be used include those that are suitable for mammalian hosts and based on viral replication systems, such as simian virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papilloma virus (BPV), papovavirus BK mutant (BKV), or mouse and human cytomegalovirus (CMV), as well as Moloney murine leukemia virus (MMLV), natural Ig promoters, etc. A variety of suitable vectors are known in the art, including vectors that are maintained in single or multiple copies, or integrated into host cell chromosomes, for example, via LTRs, or artificial chromosomes with multiple integration sites engineered (Lindenbaum et al., Nucleic Acids Res. 32: e172 (2004), Kennard et al., Biotechnol. Bioeng. 2009 May 20 online). Additional examples of suitable vectors are listed in the following sections.

Thus, the present invention provides one or more expression vectors comprising nucleic acids encoding antibodies, antigen-binding fragments of antibodies (e.g., human, humanized, chimeric antibodies, or any antigen-binding fragments of the foregoing), antibody chains (e.g., heavy chains, light chains), or antigen-binding portions of antibody chains that bind to TGFβ or TGFβR. In some embodiments, the present invention provides one or more expression vectors comprising nucleic acid extracellular domains of TGFβR.

Expression in eukaryotic host cells is useful because such cells are more likely to assemble and secrete correctly folded and immunologically active antibodies than prokaryotic cells. However, any produced antibodies that are inactive due to improper folding can be reactivated 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, pp. 459-89 (1982)). Host cells may produce a portion of a complete antibody, such as a light chain dimer or a heavy chain dimer, which is also an antibody analog according to the present invention.

The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the nucleic acid encoding the CAR construct described herein.

In one embodiment, the nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express the mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide or peptide, and the vector contacts the cell under conditions sufficient to express the mRNA, protein, polypeptide or peptide in the cell. The vector does not exist naturally as a whole.

However, a portion of the vector may be naturally occurring. The recombinant expression vector may comprise any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, 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, the 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 can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for reproduction and amplification or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of: pUC series (Fermentas Life Sciences, Glen Burnie, 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 λ, λZapII (Stratagene), EMBL4, and λNMI 149, may 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. A lentiviral vector is a vector derived from at least a portion of a lentiviral genome, particularly including self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, lentiviral vectors from Oxford BioMedica plc. Gene delivery technology, LENTIMAX ^™ vector system from Lentigen, etc. 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 coprecipitation (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 can be prepared using standard recombinant DNA techniques such as described by Sambrook et al., supra and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

The recombinant expression vector may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, where appropriate, and taking into account whether the vector is DNA-based or RNA-based, which are specific for the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced. The recombinant expression vector may contain restriction sites to facilitate cloning.

The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Marker genes suitable for expression vectors of the present invention include, for example, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector may include a natural or non-natural promoter, which is operably connected to a nucleotide sequence encoding the CAR (including its functional part and functional variant) or a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR. The selection of promoters, such as strong, weak, inducible, tissue-specific and developmentally specific, is within the ordinary skill range of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the skill range of the technician. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat sequence of a murine stem cell virus.

The recombinant expression vector can be designed for transient expression, stable expression, or both. In addition, the recombinant expression vector can be prepared for constitutive expression or inducible expression.

In addition, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cell death in which the suicide gene is expressed. A suicide gene can be a gene that imparts sensitivity to an agent (e.g., a drug) to cells expressing the gene, and causes cell death when the cell contacts or is exposed to an 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., herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

Treatment

The present invention relates to a method of treatment, comprising administering to a subject an extracellular domain of anti-TGFβ, anti-TGFβR antigen binding molecules, or TGFβR. In some embodiments, the CAR and antigen binding molecules disclosed herein can be used in methods for treating or preventing mammalian diseases. In this regard, one embodiment provides a method for treating or preventing mammalian cancer, comprising administering to a mammal an amount of CAR, nucleic acid, recombinant expression vector, host cell, cell colony, antibody and/or antigen binding portion thereof, and/or pharmaceutical composition effective for treating or preventing mammalian cancer.

In some embodiments, CAR is expressed on donor cells, and these cells secrete anti-TGFβ, anti-TGFβR antigen binding molecules or TGFβR extracellular domains. In some embodiments, donor T cells for T cell therapy are obtained from patients (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from non-patient subjects (e.g., allogeneic T cell therapy). CAR+T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells may be at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ cells or at least about 10 ¹⁰ cells.

In some embodiments, the therapeutically effective amount of T cells is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2X10 ⁶ cells/kg, about 3X10 ⁶ cells/kg, about 4X10 ⁶ cells/kg, about 5X10 ⁶ cells/kg, about 6X10 ^{6 cells/kg, about 7X10 6 cells} /kg, about 8X10 ⁶ cells/kg, about 9X10 6 cells/kg, about 1X10 ⁷ cells/kg, about 2X10 ⁷ cells/kg, about 3X10 ⁷ cells/kg, about 4X10 ⁷ cells/kg, about 5X10 ⁷ cells/kg, about 6X10 ⁷ cells/kg, about 7X10 ⁷ cells/kg, about 8X10 ⁷ cells/kg, or ^{about 9X10 7 cells} /kg. In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 1X10 ⁶ to about 2X10 ⁶ CAR-positive live T cells/kg body weight, up to a maximum dose of about 1x10 ⁸ CAR-positive live T cells.

In some embodiments, the therapeutically effective amount of live CAR-positive T cells is between about ^0.25X106 and ^2X106 . In some embodiments, the therapeutically effective amount of CAR-positive live 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} CAR-positive live T cells.

In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 0.4x10 ⁸ and about 2x10 ⁸ CAR-positive live T cells. In some embodiments, the therapeutically effective amount of CAR-positive live 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 CAR-positive live T cells.

One embodiment also includes subjecting the mammal to lymphodepletion prior to administering the CAR disclosed herein. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, whole body irradiation, and the like.

For the purpose of the method in which a host cell or cell colony is administered, the cell may be a cell that is allogeneic or autologous to the mammal. In some embodiments, the cell is autologous to the mammal. In some embodiments, the cell is allogeneic to the mammal. As used herein, allogeneic refers to any material derived from different animals of the same species as the individual into which the material is introduced. When the genes on one or more loci are not identical, the two or more individuals are referred to as allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species may be sufficiently different in genes to interact antigenically. As used herein, "autologous" refers to any material derived from the same individual, which is later reintroduced into the individual.

The mammals mentioned herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to: rodent mammals, such as mice and hamsters, and lagomorph 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 swine (pigs), or from the order Eveniodactyla, including equines (horses). The mammal may be primates, Ceboids or Simoids (monkeys) or anthropoids (humans and apes). In some embodiments, the mammal is a human.

With respect to the methods, the cancer can be any cancer, including acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder sarcoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, uterine cancer, Any of cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney 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 "treating" and "preventing" and their derivatives do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the methods can provide any amount or level of treatment or prevention of cancer in a mammal.

In addition, the treatment or prevention provided by the method may include treating or preventing one or more disorders or symptoms of the disease (e.g., cancer) to be treated or prevented. In addition, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or disorders.

The method for testing the ability of CAR to recognize target cells and antigen specificity is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999), teach the method for measuring the release of cytokines (such as interferon-γ, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)). In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, such as Zhao et al., J.Immunol, 174: 4415-4423 (2005).

Another embodiment provides the use of CAR, nucleic acid, recombinant expression vector, host cell, cell colony, antibody or antigen binding portion thereof and/or pharmaceutical composition of the present 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 can 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 can be used. The specific 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, disease state involved, and whether the treatment is preventive). In the case of administering more than one agent or composition, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen binding fragments or conjugates or compositions may be administered intravenously. The method of administration includes injection, wherein CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in non-toxic pharmaceutically acceptable carriers such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, non-volatile oils, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying antibodies or antigen binding fragments to tissue areas where tumors have been removed, or areas suspected of being prone to developing tumors. 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 eye drops, or intravitreally to the eye.

The disclosed therapeutic agents can be formulated into unit dosage forms suitable for single administration of precise doses. In addition, the disclosed therapeutic agents can be administered in single doses or multiple dose regimens. Multiple dose regimens are where the main course of treatment may be more than one single dose, for example 1 to 10 doses, followed by other doses administered at subsequent time intervals as needed to maintain or enhance 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. Therefore, the dosing regimen will also be determined at least in part based on the specific needs of the subject to be treated and will depend on the judgment of the administering physician.

A typical dosage range for the antibody or conjugate may be about 0.01 to about 30 mg/kg, such as about 0.1 to about 10 mg/kg.

In a specific example, a therapeutic composition including one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or an additional agent is administered to a subject with a multiple daily dosing regimen, e.g., for at least two consecutive days, for 10 consecutive days, etc., e.g., for weeks, months, or years. In one example, a conjugate, an antibody, a composition, or an additional agent is administered to a 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 method includes providing surgery, radiotherapy and/or chemotherapeutic agents to the subject, in combination with the disclosed antibodies, antigen binding fragments, conjugates, CARs or T cells expressing CARs (e.g., sequentially, substantially simultaneously or simultaneously). Such agents and treatment methods and therapeutic doses are known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosing regimen of additional agents can be determined empirically according to the manufacturer's instructions or by skilled professionals. The preparation and dosing regimen of such chemotherapy is also described in Chemotherapy Service, (1992), M.C.Perry, ed., Williams & Wilkins, Baltimore, Md.

In some embodiments, the combination therapy may include administering to the subject an additional cancer inhibitor in a therapeutically effective amount. Non-limiting examples of additional therapeutic agents that may be used with the combination therapy include microtubule binders, DNA chimeras or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered in a therapeutically effective amount) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by skilled clinicians.

Other chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemtuzumab; gemcitabine; plant alkaloids, such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca roseus (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, epirubicin, mitoxantrone, topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and trastuzumab. sodium, and verteporfin; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukindiftitox, erlotinib, estramustine, The present invention relates to sirolimus, gefitinib, hydroxyformamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib and tretinoin. The selection and therapeutic dosage of such agents are known to those skilled in the art and can be determined by a skilled clinician.

The combination therapy may provide a synergistic effect and demonstrate synergy, i.e., an effect when the active ingredients are used together that results in an effect greater than the sum of the effects of the compounds when used separately. A synergistic effect may be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) delivered as separate formulations in alternation or in parallel; or (3) by some other regimen. When delivered in an alternating manner, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, e.g., by different injections in different syringes. Generally, during alternation, an effective dose of each active ingredient is administered sequentially, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

In various embodiments, the immunomodulatory system comprising a TGFβ signaling modulator as described herein may be included in a treatment process that also includes administering at least one additional agent to the subject. In various embodiments, the additional agent administered in combination with the immunomodulatory system comprising a TGFβ signaling modulator 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 regulator is a single domain antibody or a secretable scFv specific for human TGFβ. In some embodiments, the TGFβ signaling regulator is a single domain antibody or a secretable scFv specific for human TGFβR. In some embodiments, the TGFβ signaling regulator can be conjugated (e.g., connected) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) to bind to cancer cells, deliver the therapeutic agent to cancer cells, and kill cancer cells expressing human TGFβ. In some embodiments, the TGFβ signaling regulator is connected to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a cytokine, a radioactive atom, an siRNA, or a toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the agent is a radioactive atom.

In some embodiments, the method can be combined with other therapies for abnormal TGFβ signaling disorders. For example, the composition can be administered to the subject simultaneously before or after chemotherapy. In some embodiments, the composition can be administered to the subject simultaneously, before or after adoptive cell therapy.

In various embodiments, the additional agent administered in combination with the immunomodulatory system comprising the TGFβ signaling regulator described herein may be administered at the same time, on the same day, or in the same week as the TGFβ signaling regulator. In various embodiments, the additional agent administered in combination with the TGFβ signaling regulator described herein may be administered together with the immunomodulatory system in the form of a single formulation. In certain embodiments, the additional agent is administered in a temporally separate manner from the administration of the TGFβ signaling regulator as described herein, for example, one or more hours before or after the administration of the TGFβ signaling regulator, one or more days before or after, one or more weeks before or after, one or more months before or after. 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 the TGFβ signaling regulator as described herein.

In some embodiments, the composition may be formulated with one or more additional therapeutic agents, such as additional therapies for treating or preventing a TGFβ-related disorder (e.g., cancer or an autoimmune disorder) in a subject. The additional agents used to treat a TGFβ-related disorder in a subject will vary depending on the specific disorder to be treated, but may include, but are not limited to, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, ifosfamide, carboplatin, etoposide, dexamethasone, cytarabine, cisplatin, cyclophosphamide, or fludarabine.

Composition

Provided herein are compositions for gene therapy, immunotherapy and/or cell therapy, which include one or more disclosed CARs or T cells expressing CAR, antibodies, antigen binding fragments, conjugates, CARs or T cells expressing CAR (which specifically bind to one or more antigens disclosed herein) in a carrier (e.g., a pharmaceutically acceptable carrier). The composition can be prepared for a unit dosage form administered to a subject. The dosage and time of administration are determined by the attending clinician at his discretion to achieve the desired results. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CAR or T cells expressing CAR, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions including CARs or T cells expressing CAR, conjugates, antibodies, or antigen binding fragments as disclosed herein can be used, for example, to treat and detect tumors, such as and not limited to neuroblastoma. In some examples, the composition can be used to treat or detect cancer. Compositions comprising a CAR or CAR-expressing T cell, conjugate, antibody or antigen-binding fragment as disclosed herein are also used, for example, to detect pathological angiogenesis.

The composition for administration may include a solution of CAR or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers may be used, for example, buffered physiological saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR or T cells expressing CAR, antibodies or antigen binding fragments or conjugates in these preparations may vary widely, and will be selected mainly according to the selected specific mode of administration and the needs of the subject, mainly based on fluid volume, viscosity, body weight, etc. The actual method of preparing such dosage forms for gene therapy, immunotherapy and/or cell therapy is known or will be obvious to those skilled in the art.

Typical compositions for intravenous administration include about 0.01 to about 30 mg/kg of antibody or antigen-binding fragment or conjugate (or corresponding doses of CAR, or T cells expressing CAR, or conjugates including antibody or antigen-binding fragments) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, Pa. (1995).

Controlled release parenteral formulations can be made into implants, oily injections, or 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 therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, so only nanoparticles can be administered intravenously. Microparticles are generally about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339 (1992).

Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or antigen binding fragments or conjugate compositions for ion controlled release. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer polaxamer 407 exists in a viscous but flowing liquid form at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective medium for the preparation 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 proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). In yet another aspect, liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). A variety of additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Pat. 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).

Pill Box

In one aspect, a kit using the CAR disclosed herein is also provided. For example, a kit for treating a tumor in a subject, or for making a CAR T cell expressing one or more CARs disclosed herein. The kit will generally include an antibody, an AF, a conjugate, a nucleic acid molecule, a CAR, or a T cell expressing CAR as disclosed herein. More than one disclosed antibody, AF, a conjugate, a nucleic acid molecule, a CAR, or a T cell expressing CAR may be included in the kit.

The medicine box may include a container and a label or package insert on or connected to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of various materials such as glass or plastic. The container generally holds a composition, which comprises one or more of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. 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 that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific disease.

The label or package insert will generally further include instructions for use of the disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cell, for example, for treating or preventing tumors or manufacturing CAR T cells. The package insert generally includes instructions conventionally contained in the commercial packaging of the therapeutic product, which contain information about the indications, usage, dosage, administration, contraindications and/or warnings for the use of such therapeutic products. The instruction material may be written, in electronic form (e.g., computer hard disk or CD) or visual form (e.g., visual file). The medicine box may also include additional components to facilitate specific applications of the medicine box design. Therefore, for example, the medicine box may additionally contain a device for detecting a marker (e.g., an enzyme substrate for an enzyme marker, a filter group for detecting a fluorescent marker, a suitable secondary marker (e.g., a secondary antibody), etc.). The medicine box may additionally include a buffer and other reagents conventionally used to implement a specific method. Such medicine boxes and appropriate contents are well known to those skilled in the art.

Unless otherwise indicated, all technical and scientific terms and phrases used herein have the same meaning as those generally understood by those of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, 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). Enzyme reactions and purification techniques can be performed according to the manufacturer's instructions or as generally completed in the art or as described herein. The aforementioned techniques and procedures can generally be performed according to conventional methods described in various general and more specific references as well known in the art and cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd 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 present invention will be more fully understood by reference to the following examples.

Example

These examples are presented to help understand the present invention, but are not intended to and should not be interpreted as limiting its scope in any way. The examples do not include a detailed description of conventional methods known to those of ordinary skill in the art (molecular cloning techniques, etc.).

Example 1. Immunoreactive cells co-expressing chimeric antigen receptor (CAR) and TGF-B signaling regulator

This example illustrates the use of an immunomodulatory system to co-express TGF-β signaling regulators and CARs in human T cells. The immunomodulatory constructs encoding TGF-B signaling regulators (e.g., anti-TGFβ and anti-TGFβR2) and anti-human CD19 CAR (SJ25C1 extracellular antigen binding domain) are packaged for retroviral delivery. Phoenix A retroviral packaging cell line (ATCC) is grown to 50-70% confluence in DMEM 20% FBS and penicillin/streptomycin. According to the manufacturer's protocol, DNA complexes are prepared using each plasmid encoding TGF-B signaling regulator and CAR construct, auxiliary plasmid gag-pol, and pVSVG and transduction reagent Fugene HD (Promega). After transfection for 20-48 hours, the viral supernatant is collected, aliquoted and frozen for further use.

Human PBMCs were isolated by Leukopaks using a density gradient and frozen until further use. Human T cells were isolated by magnetic selection from previously frozen PBMCs (T cell isolation kit; Stemcell). Purified human T cells were cultured for 2 days in a complete Optimizer culture medium (Optimizer basal culture medium (ThermoFisher#A10221-01) + 26ml OptiMizer supplement (ThermoFisher#A10484-02) + 20ml ICSR (CTS immune cell SR), ThermoFisher#A25961-01) + 10ml of 200mM L-glutamine, (Gibco25030-081) + 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 rotated with an appropriate amount of virus. Transduction was confirmed and quantified by flow cytometry at different time points. In brief, cells were incubated with 250ng hCD19-hFc protein (RnD Systems) or internally produced hGCC-Fc protein at 4'C in FACS buffer for 1 hour. After washing with FACS buffer, the cells were resuspended in a secondary antibody (Biolegend) against human FC for 20 minutes at room temperature. Antibodies against CD4, CD8 or other surface markers were added in some experiments. Dead cells were excluded from the analysis using a fixable viability dye (Thermofisher). Before analysis by flow cytometry (FACSFortessa, BD Biosciences), cells were fixed in PBS2% FCS 4% formaldehyde. Transduction efficiency is shown as the percentage of live cells positive for CAR staining. Flow cytometry results showed that 87.6% of lymphocytes, 76.1% of single cells, 78.3% of live CD3+ cells, and 75.8% of the population of cells showed CAR expression (Figures 1A-1D). Transduction efficiency was shown as the percentage of live cells that stained positive for CAR (Figure 1E).

Example 2. In vitro killing using TGF-β-regulated human CAR-T cells

This example demonstrates in vitro killing by human CAR-T cells co-expressing a TGF-β signaling regulator. The in vitro killing of armored human CAR-T cells was comparable to that of unarmored CAR-T cells.

Raji (ATCC CD19 positive) or Raji CD19ko (human CD19 negative) was stained with the proliferation dye efluor 450 (Thermofisher) and plated in a 96-well plate for at least 2 hours according to the manufacturer's protocol, after which the TGF-β regulated CAR-T cells described in Example 1 were added. CAR-T cells were added with only T cells at a ratio of 0:1, 0.3:1, 1:1, 3:1, 9:1 for effector: target and incubated overnight at 37°C. The next day, FACS staining was performed using fluorescent dye-conjugated antibodies against human CD107a (LAMP-1) (Biolegend), TCRα/β (Biolegend), and human CD4 antibodies (Biolegend). According to the manufacturer's protocol, the cells were incubated with antibodies at 4°C for 30 minutes, washed with PBS, and stained with a fixable viability dye (Thermofisher). The cells were washed with 1x Annexin V binding buffer (Biolegend) and stained with Annexin V FITC. The cells were fixed in cytofix (BD Biosciences) and then acquired on FACSFortessa (BD Biosciences). TGF-β-regulated CD19 CAR-T cells exhibited target-specific in vitro killing of CD19-positive Raji cells (Figure 2A), but not CD19-negative control cells (Raji CD19ko) (Figure 2B).

Example 3. Secretion of TGF-β modulators by immunoreactive cells

This example demonstrates that TGF-β-regulated CAR-T cells secrete co-expressed TGF-β modulators (e.g., anti-TGF-β that binds to TGF-β and anti-TGFβR2 that binds to TGFβR2).

Supernatants from TGF-β-regulated CAR-T cells were assayed by ELISA to detect anti-TGF-β and anti-TGFβR2 antibodies. Maxisorp 96-well plates were coated overnight with recombinant human TGF-β (4; RnD System μg/ml) or hTGFβR2-Fc (0.1 mg/ml; RnD System) in 100 μl coating buffer at 4°C. The plates were washed with 1x wash buffer and blocked with reagent diluent for 1 hour at room temperature. CAR-T supernatants, recombinant TGFβR2-flag, or recombinant TGF-β-flag antibodies were added and incubated for 2 hours at room temperature.

After another washing step, add the flag tag antibody conjugated with HRP and incubate at room temperature for 30 minutes.The plate is washed and TMB substrate is added for 10-20 minutes.The reaction is terminated using a stop reagent and the plate is read at 450nm using a Pherastar plate reader.Compared with using anti-flag tag HRP antibodies for binding sub-detection with TGF-b scFv VL-VH, high levels of TGF-b scFv VH-VL1 and TGF-b scFv VH-VL2 (Fig. 3A) were detected using the ELISA of the TGF-b coated.High levels of TGFbR2 scFv VH-VL, TGFbR2scFv VL-VH and hTGFbR2 VH1 from human CAR-T were detected using the ELISA of the TGFbR2-Fc coated, but mTGFbR2 VH1 (Fig. 3B) could not be detected. TGF-β binder and TGFβR2 binder are secreted by TGF-β regulated CAR-T cells and bind to their cognate antigens.

Example 4. Human CAR-T cells secrete neutralizing antibodies against TGF-β/TGFβR2

This example demonstrates the presence of neutralizing antibodies against TGF-β/TGFβR2 in the supernatant of TGF-β-regulated CAR-T cells.

Functional evaluation of TGF-β blocking binders in CAR-T cell supernatants was performed using SBE-Luc reporter cells (HEK293 cells expressing firefly luciferase under the control of Smad binding elements (SBE) (BPS Biosciences), which are designed to monitor the activity of the TGF-β/SMAD signaling pathway. The TGF-β protein binds to its cognate receptor on the cell surface, triggering a signal transduction cascade that leads to phosphorylation and activation of SMAD2 and SMAD3, and then forms a complex with SMAD4. The SMAD complex is translocated to the nucleus and binds to the SMAD binding element (SBE), resulting in the transcription and expression of TGF-β/SMAD responsive genes. The presence of blocking binders is detected by its ability to inhibit the expression of TGF-β inducible luciferase in SBE-Luc reporter cells. An exemplary assay for evaluating the efficacy of inhibiting TGF-β inducible reporter activity is performed as follows.

SBE-Luc cells were seeded into 96-well plates coated with poly-D-lysine at a concentration of 1x10 ⁵ cells in 100 μl of fresh medium (X-VIVO15 containing 1x penicillin/streptomycin) per well and incubated for 4 hours at 37°C and 5% CO _2. The supernatant from CAR-T cells or its dilution was mixed with an equal volume of TGF-β (4 ng/ml in X-VIVO15) and incubated at room temperature for 15 minutes to allow TGF-β to complex with TGF-b contained in the CAR-T supernatant. 100 μl of the mixture was added to the SBE-Luc reporter cells in duplicate and incubated overnight at 37°C and 5% CO _2. The final concentration of TGF-β was 1 ng/ml. Each experiment included a titration curve of increasing dilutions of TGF-β antibody (1D11 (BioXcell) or TGF-β binder or TGFβR2 binder) in the presence of 1 ng/ml TGF-β.

On the second day, 100 μl of culture supernatant was removed and 100 μl of detection reagent (ONE-Step ^TM luciferase assay system) containing Luciferin-D was added. Cells were resuspended and transferred to a white detection plate and luminescence was measured using a Pherastar plate reader. Luciferase activity was recorded in CPM. Data were analyzed using MS Excel or Graphpad prism. Nonlinear regression fitting was performed using the S-shaped dose-response (variable slope) of Graphpad prism. IC50 values were calculated.

The inhibitory activity (%) was calculated using the following equation:

Inhibition (%) = (1-CPM of sample/maximum CPM of sample treated with TGF-β (1 ng/ml)) X 100

The results showed that the supernatant from the CAR-T cells of the secretory constructs TGF-βscFv VH-VL1 (SEQ ID NO: 1) and TGF-βscFv VH-VL2 (SEQ ID NO: 2) inhibited TGF-β signaling (Fig. 4). Additional constructs were designed and luciferase reporter assays were used to screen the secretion of multimeric binders of anti-TGF-β or TGFβR2 (Fig. 5 and Fig. 6). TGF-β regulated CAR-T cells that secrete multimeric antibodies against TGF-β and TGFβR2 were identified. Regardless of the joint, human CAR-T cells can secrete multimeric TGF-b binders and inhibit TGF-b signaling. As shown in the figure below, four different joints were analyzed. Similar results were observed using anti-GCC CAR-T cells (data not shown).

Example 5. TGF-β-regulated CAR-T cells secrete multimeric binders against TGF-β or TGFβR2

This example illustrates the screening and identification of mouse CAR-T cells that secrete multimeric binders of anti-TGF-β or TGFβR2. In order to produce mouse CAR-T cells, Platinum-E retroviral packaging cell lines were grown to 50-70% confluence in DMEM 20% FBS and penicillin/streptomycin. According to the manufacturer's protocol, DNA complexes were prepared using immunomodulatory system plasmids encoding CAR constructs and TGFB regulators (e.g., anti-TGF-b scFv monomers, anti-TGF-b scFv dimers), packaging constructs, and transduction reagents Fugene HD. The solution was mixed and incubated at room temperature for 10 minutes, and 850 μl of the complex was added to each 10 cm ² cell dish. Mouse T cells were isolated from Balb/c or C57BL/6 mouse spleens using a T cell separation kit with magnetic selection. Purified mouse T cells were cultured with mouse T cell activation beads (1:1 ratio) in RPMI 10% heat-inactivated FCS, penicillin/streptomycin and mouse IL-2 (30U/ml) for 2 days. Virus was collected approximately 48 hours after transfection and filtered through a 0.4μm syringe filter. T cells were transferred to plates coated with retronectin (40μg/ml retronectin according to the manufacturer's protocol) and spin-transduced with appropriate viral amounts. Transduction was confirmed and quantified at different time points by flow cytometry.

The cells were incubated with 250ng hCD19-hFc protein in FACS buffer for 1 hour at 4°C. After washing with FACS buffer, the cells were resuspended with a secondary antibody against human FC for 20 minutes at room temperature. In some experiments, antibodies against CD4, CD8 or other surface markers were added. Dead cells were excluded from the analysis using a fixable viability dye. Before analysis by flow cytometry (FACS Fortessa), the cells were fixed in PBS 2% FCS, 4% formaldehyde. Transduction efficiency is shown as the percentage (%) of live cells that are positive for CAR staining.

Flow cytometry results show the relative proportions of unarmored T cells, TGF-β monomers, TGF-β dimers, and untransduced cells. Supernatants from mouse CAR-T cells collected at d+2 after transduction were probed for TGF-b signaling inhibition in the SBE-Luc TGF-b reporter assay (as described in Example 4). Based on luciferase reporter activity, supernatants from mouse CAR-Ts that secrete 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 after transduction and frozen at -80'C until used for ELISA. ELISA was performed as described in Example 3. Human recombinant TGFbR2-Fc protein was used to capture the binder of anti-human TGFbR2, and mouse recombinant TGFbR2-Fc protein was used to detect the binding of TGFbR2 binders to mouse TGFbR2. Anti-flag HRP antibody and individual substrates were used to detect binding. As shown in Figure 7, the secretion of binders hTGFbR2-VH2 and hTGFbR2-VH3 monomers and dimers and monomers and dimers of TGFbR2 scFv VH-VL bind to human TGFbR2. None of the binders in the tested supernatants bind to mouse TGFbR2, confirming the specificity for human TGFBR2.

Example 6. In vivo anti-tumor efficacy of CAR-T cells secreting TGF-β signaling regulators

This example illustrates the in vivo anti-tumor efficacy of CAR-T cells secreting anti-TGF-βmAb. Mouse armored CAR-T cells (co-expressing anti-human CD19 CAR and TGFb signaling regulator) inhibited the growth of isogenic EMT6-hCD19 tumors better than unarmored CAR-T cells. In addition, armored CAR-T cells reduced liver and lung metastasis. EMT6 breast cancer cell lines overexpressing human CD19 and firefly luciferase as CAR-T target antigens were generated. EMT6 cells were transduced with a plasmid encoding human CD19 under the control of the EF1a promoter and a virus with puromycin resistance. EMT6-hCD19 cells were positively selected using puromycin and further purified by FACS sorting. EMT6-hCD19 cells were transduced with a virus carrying a plasmid encoding firefly luciferase under the control of the EF1a promoter and conferring neomycin resistance (Amsbio) at ^5x107 IFU/mL in the presence of polybrene; MOI = 10. EMT6-hCD19-Fluc cells were positively selected using G418 (500 μg/ml).

0.2x10 ⁶ live EMT6-hCD19-Fluc tumor cells were inoculated into the mammary fat pad (orthotopic) of 6-16 week-old female Balb/c mice (Jackson Labs). After 6 days of implantation, the tumor size reached about 50mm ³ and the mice were randomly grouped into treatment groups with similar average tumor size (average about 50mm ³ ) and treated with cyclophosphamide (CPA; 200mg/kg i.p.). The next day, 500,000 mouse CAR-T cells from congenic CD45.1 Balb/c mice were injected into the tail vein. Group 1 received untransduced T cells, Group 2 received CAR-T cells, and Group 3 received CAR-T cells secreting anti-TGF-βscFvVH-VL1. Body weight was measured twice a week to monitor toxicity.

Tumor size was measured twice a week and tumor volume was calculated using the following formula: Tumor volume (mm ³ ) = length x width x height x 0.5236 ( FIG. 8 ). Any mouse with a tumor exceeding 2000 mm ³ or with an ulcerated tumor was sacrificed. Antitumor efficacy was evaluated as a reduction in tumor size compared to control mice injected with non-transduced T cells. Complete responders were defined as mice without any detectable tumor.

Relative to unarmored CAR-T or untransduced CAR-T cells, CAR-T cells that secrete TGF-β binders show high anti-tumor efficacy. Tumor cells expressing firefly luciferase in the liver and lungs were imaged by injecting luciferin and imaging by IVIS. D-luciferin solution (D-luciferin, Vivo Glo ^TM luciferin potassium salt) was prepared as 15mg/ml and used at 150mg/kg. Mice treated with CAR-T cells that secrete TGF-β binders were able to reduce metastasis in the liver and lungs (Figures 8A-8E).

Mouse CAR-T cells against human CD19 that secrete inhibitory antibodies against TGF-β (TGF-b scFv VH-VL1) inhibited syngeneic EMT6-hCD19 tumor growth better than unarmored CAR-T cells and reduced liver and lung metastasis. The number of CAR-T cells had been titrated previously to obtain suboptimal effects for unarmored CAR-T to identify the improved activity of armored CAR-T cells.

Example 7. Armored mouse CAR-T cells secreting the extracellular domain (ECD) of TGFbR2 inhibit TGFb signaling guide

A SBE-Luc TGF-β reporter assay was performed comparing supernatants from armored mouse CAR-Ts secreting different TGF-β ligand traps (TGF-βscFv VH-VL1 to TGFbR2 ECD monomers, homodimers (Figure 9A), and heterodimers (Figure 9B)) to supernatants from unarmored CAR-Ts. The SBE-Luc TGF-β reporter assay showed good inhibitory effects from supernatants from anti-human CD19 armored mouse CAR-Ts secreting TGFβR2 extracellular domain (ECD) dimers (but not monomers), and comparable inhibitory effects from those secreting TGF-βscFv VH-VL1 dimers. Supernatants were collected 2 days after transduction. TGFβR2 ECD heterodimers including both TGFβR2 ECD and TGFβR1 ECD were evaluated for inhibitory effects. TGFβR2 ECD heterodimers were identified that inhibited TGF-β signaling more effectively than TGF-β scFV VH-VL1. Exemplary TGFβR2 ECD sequences are shown in Table 4.

Table 4. Exemplary TGFβR2 ECD sequences

Example 8. Anti-tumor efficacy of armored CAR-T cells secreting TGF-β signaling regulators

This example illustrates the relative anti-tumor efficacy of CAR-T cells secreting anti-TGFβ mAb or TGFβR2-ECD in vivo.

Compared with unarmored CAR-T cells, mouse CAR-Ts that secrete TGFβR2 ECD1+2 dimers show improved anti-tumor effects in vivo. 0.2x10 ⁶ live EMT6-hCD19-Fluc tumor cells were inoculated into the mammary fat pad (orthotopic) of 6-16 week-old female Balb/c mice (Jackson Labs). After 6 days of implantation, the tumor size reached about 50mm3 and the mice were randomly grouped into treatment groups with similar average tumor sizes (average of about 50mm3; n=8 per group) and treated with cyclophosphamide (CPA; 200mg/kg ip). The next day, 2 million mouse CAR-T cells from congenic CD45.1 Balb/c mice were injected into the tail vein. Group 1 received untransduced control T cells, Group 2 received unarmored CAR-T cells, Group 3 received CAR-T cells secreting TGFbR1+2ECD dimers, and Group 4 received systemic anti-TGF-β antibody (clone 1D11.16.8; 10 mg/kg; 3 injections per week; iv). Body weight was measured twice a week to monitor toxicity. Tumor size was measured twice a week and tumor volume was calculated using the following formula: Tumor volume (mm ³ ) = length x width x height x 0.5236. Following the association's animal health protocol, mice with any tumors exceeding 2000 mm ³ or with ulcerated tumors were sacrificed. Antitumor efficacy was evaluated by a reduction in tumor size compared to control mice injected with untransduced T cells. Complete responders were defined as mice without any detectable tumors.

Anti-human CD19 mouse CAR-Ts secreting the TGF-β ligand trap (mTGFbR2 ECD1+2 dimer 1) suppressed syngeneic EMT6-hCD19 tumor growth better than unarmored CAR-T cells, inducing 3 complete responses compared to no complete responses in control mice receiving unarmored CAR-Ts or untransduced T cells, or treated with systemic anti-TGF-β antibodies (1D11, 10 mg/kg, 3x i.v. per week) (Figure 10).

Example 9. Armored CAR-T cells secreting TGF-β signaling regulators in a syngeneic tumor model Anti-tumor efficacy in (MC38-hCD19)

This example illustrates the relative anti-tumor efficacy of CAR-T cells secreting anti-TGFβmAb or TGFβR2-ECD in vivo. Mouse CAR-T cells armored with anti-TGF-b mAb (TGF-b scFv VH-VL1) had improved effects in different syngeneic tumor models (MC38-hCD19).

MC38 colorectal cancer cell lines overexpressing human CD19 and firefly luciferase as CAR-T target antigens were generated and used for imaging. In brief, MC38 cells were transduced with a plasmid encoding human CD19 under the control of the EF1a promoter and a virus with puromycin resistance (CD19_FL_WT_pLVX-EF1a-IRES-Puro). MC38-hCD19 cells were positively selected using puromycin. MC38-hCD19 cells were transduced with a plasmid encoding firefly luciferase under the control of the EF1a promoter and a virus with neomycin resistance (Amsbio, catalog number LVP435-PBS, with 5x10^7IFU/mL, MOI=10) in the presence of polybrene. MC38-hCD19-Fluc cells were positively selected using G418 (Geneticin).

6-16 week old female C57BL/6 mice (Jackson Labs) were subcutaneously inoculated with 0.2x10 ⁶ live MC38-hCD19-Fluc tumor cells. Seven days after implantation, the tumor size reached about 50mm3 and the mice were randomly grouped into treatment groups with similar average tumor sizes (average of about 50mm3; n = 8 per group) and treated with cyclophosphamide (CPA; 200mg/kg ip). The next day, 100,000 mouse CAR-T cells (or untransduced T cells as negative controls) from congenic CD45.1 C57BL/6 mice were injected into the tail vein. Group 1 received untransduced T cells. Group 2 received unarmored CAR-T cells. Group 3 received CAR-T cells (TGF-b scFv VH-VL1) that secrete anti-TGF-β. Body weight was measured twice a week to monitor toxicity. Tumor size was measured twice a week and tumor volume was calculated using the following formula: Tumor volume (mm ³ ) = length x width x height x 0.5236. Following the Institute's animal health protocol, mice with any tumor exceeding 2000 mm ³ or with ulcerated tumors were sacrificed. Antitumor efficacy was evaluated as a reduction in tumor size compared to control mice injected with non-transduced T cells. Complete responders were defined as mice without any detectable tumor.

CAR-T cells secreting an inhibitory binder against TGF-β (TGF-b scFv VH-VL1) showed superior efficacy to unarmored CAR-T, inducing 7 complete responses in 8 treated mice compared to no complete responses in the control group that received an equal amount of unarmored CAR-T or untransduced T cells (Figure 11).

Example 10. CAR-T cells secreting TGF-β signaling regulators enhance the activation of host immune responses

This example presents RNA Seq showing that the host immune response is enhanced by activation of CAR-T cells that secrete anti-TGF-β binders.

0.2x10 ⁶ live EMT6-hCD19-Fluc tumor cells were inoculated into the mammary fat pad (orthotopic) of 6-16 week-old female Balb/c mice (Jackson Labs). After 6 days of implantation, the tumor size reached about 50mm3 and the mice were randomly grouped into treatment groups with similar average tumor sizes (average of about 50mm3; n = 8 per group) and treated with cyclophosphamide (CPA; 200mg/kg ip). The next day, 2 million mouse CAR-T cells from congenic CD45.1 Balb/c mice were injected into the tail vein. Group 1 received untransduced control T cells, Group 2 received unarmored CAR-T cells, and Group 3 received CAR-T cells secreting anti-TGF-βscFv VH-VL1. Group 4 was treated with systemic anti-TGF-β antibody (clone 1D11.16.8; BioXcell; 10 mg/kg; 3 times per week 1.v.), and Group 5 received an isotype control antibody (clone MOPC21; BioXcell; 10 mg/kg; 3 times per week 1.v.). Body weight was measured twice per week to monitor toxicity. Tumor size was measured twice per week and tumor volume was calculated using the following formula: Tumor volume (mm ³ ) = length x width x height x 0.5236.

Mice were euthanized on day +12 and tumors were harvested, snap frozen and kept at -80° C. RNA was extracted and subjected to RNA-Seq and subsequent computational analysis as shown in FIG12 .

Compared with mice from other groups, tumors from mice receiving CAR-T cells secreting TGF-βscFv VH-VL1 had significantly increased scores of tumor-infiltrating T cells (CD3d+, CD3e+, CD3g+), especially CD8+ T cells (CD8a+) and cell-killing T cells (GzmB+) (Figure 13)

ssGSEA enrichment scores showed an increase in T cell signatures and IFNg signatures in mouse tumors from CAR-Ts that secreted TGF-βcFv VH-VL1, indicating increased infiltration of CAR-T cells and/or increased activation of the endogenous immune system. The increased signatures of activated endothelial cells, co-stimulation, and antigen presentation in mouse tumors from CAR-Ts that secreted TGF-βscFv VH-VL1 clearly showed activation of the endogenous immune system. Therefore, armoring CAR-T cells with blocking antibodies (or other binders) inhibited the anti-tumor efficacy of the TGF-β pathway, at least in part by improving endogenous immune responses. (Figure 14).

Example 11. FACS of tumor samples from EMT6-hCD19 mice treated with unarmored CAR-T cells

0.2x10 ⁶ live EMT6-hCD19-Fluc tumor cells were inoculated into the mammary fat pad (orthotopic) of 6-16 week old female Balb/c mice (Jackson Labs). Six days after implantation, the tumor size reached about 50 mm3 and the mice were randomly grouped into treatment groups with similar average tumor size (average about 50 mm3; n = 8 per group) and treated with cyclophosphamide (CPA; 200 mg/kg ip). The next day, 2 million mouse CAR-T cells from congenic CD45.1 Balb/c mice were injected into the tail vein. Group 1 received untransduced control T cells, Group 2 received unarmored CAR-T cells, and Group 3 received CAR-T cells secreting anti-TGF-b scFv VH-VL1 monomers. Body weight was measured twice a week to monitor toxicity. Tumor size was measured twice a week 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 subjected to FACS analysis. Briefly, tumors were cut into small pieces and digested using a mouse tumor dissociation kit (Miltenyi) according to the manufacturer's instructions. Samples were resuspended in PBS 2% FCS, filtered and seeded into 96-well plates for FACS staining. Fc receptors were blocked (TruStain FcX (anti-mouse CD16/32) antibody; Biolegend) and CAR was labeled with rhuCD19 (RnD Systems) for 1 hour at 4'C, followed by hCD19-Fc labeled with anti-human IgG Fc antibody, surface markers including TCRa/b, CD8a, CD4, CD25, CD62L, CD11b, Gr1, CD11c, CD45.1 and CD45, live cells were stained with a fixable viability dye (ebioscience), and intracellular antigens including GzmB, Ki67 and FoxP3 were stained using eBioscience Foxp3/transcription factor staining buffer set (Thermofisher). Samples were filtered and acquired on a BD Fortessa flow cytometer.

As shown in Figure 15, compared with the control group receiving untransduced cells or unarmored CAR-T, FACS staining showed that the hCD19+ tumor cells in the samples of mice receiving CAR-T secreting TGF-βscFv VH-VL were reduced, and T cell infiltration (per mg tumor tissue) increased. Gating CD45.1+ and CD45.1-T cells showed that endogenous T cell infiltration (CD45.1-) was particularly increased. The CAR expression level of the transferred CAR-T cells (CD45.1+) from these samples was higher, and CD8+T cells showed higher CD25 expression, indicating increased activation. GzmB expression of CD8+T cells from host T cells (CD45.1-) was higher, indicating that cytotoxicity was higher. In summary, these FACS data show that armoring CAR-T cells with anti-TGF-β binders enhances the effect of CAR-T cells and endogenous immune responses.

Example 12. Xenograft Model Demonstrates Human GCC-1 Armored with Anti-TGF-β or Anti-TGFβR2 Blocking Antibodies Improved effect of CAR-T cells

6-16 week old female NSG mice (Jackson Labs) were subcutaneously inoculated with 2x10 ⁶ live GSU tumor cells. After 7 days of implantation, the tumor size reached about 50mm3 and the mice were randomly divided into treatment groups with similar average tumor sizes (average of about 50mm3; n = 6 per group). The next day, 500,000 or 100,000 human GCC CAR-T cells were injected into the tail vein. Group 1 received untransduced control T cells, Group 2 received unarmored CAR-T cells, Group 3 received CAR-T cells secreting anti-TGF-b scFvVH-VL1 monomers, Group 4 received CAR T cells secreting anti-TGFβR2 VH3 monomers, and Group 5 received CAR T cells secreting anti-TGFβR2 VHH dimers. Body weight was measured twice a week to monitor toxicity. Tumor size was measured twice a week and tumor volume was calculated using the following formula: Tumor volume (mm ³ ) = length x width x height x 0.5236. GCC-CAR-T cells armored with anti-TGF-β or anti-TGFβR2 blocking antibodies showed a faster response than unarmored control CAR-T when 500,000 cells were transferred, and the anti-tumor efficacy was improved when 100,000 CAR-T cells were transferred. ( FIG. 16 ).

Tumor and plasma concentrations of TGFβ modulators were determined using anti-Flag immunocapture LC/MS assays. As shown in Figures 17A-17D, small amounts of TGF-β antibodies or anti-TGFbR2 antibodies were secreted in the circulation of mice treated with armored CAR-T cells. Plasma was collected from mice treated with the indicated amounts of armored or unarmored anti-GCC CAR-T cells using EDTA tubes.

As shown in Figures 20A-20C, 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 evaluated using intrasplenic injection of HT55 tumor cells followed by intravenous injection of CAR-T cells. Armored CAR-T cells slowed metastasis to the liver relative to isotype controls (Figures 21A-21C)

Example 13. Repeated Antigenic Stimulation in GCC-Positive Tumors

100,000 anti-GCC CAR-T cells that were unarmored or armored (co-expressing anti-GCC CAR and TGFβ regulators (e.g., TGFβR2-VHH)) were co-cultured with 200,000 HT29-GCC or HT29 parent (GCC negative) tumor cells in duplicate in the presence or absence of TGF-β (1ng/ml or 10ng/ml). Half of the CAR-T cells in each well were transferred to a new tumor cell plate (with or without TGF-β1ng/ml or 10ng/ml) under the same conditions every 3-4 days. The supernatant was collected and frozen for later evaluation. Cell counting and FACS staining analysis of the evaluation cells were performed.

Tumor cells were assessed using CellTiterGlo (Promega) according to the manufacturer's protocol.

Analyze plates using a Pherastar plate reader. Estimate percent killing using the following formula:

Killing % = (1-(test well signal/control well signal))*100

Control wells contain tumor cells co-cultured with untransduced T cells from the same donor (for CAR-T cells). FACS staining was performed weekly using fluorescent dye-conjugated antibodies against human CD4, CD8, CD25 and exhaustion markers PD-1, TIM-3, Lag-3, and TIGIT antibodies (Biolegend). Dead cells were excluded using the fixable viability dye efluor 506 (Thermofisher; according to the manufacturer's protocol). CAR-expressing cells were incubated with GCC-hFc for 1 hour at 4'C, washed with PBS2% FCS and detected with a secondary mouse anti-human IgG antibody (30 minutes, 4'C).

After several rounds of restimulation with target cells (simulating chronic antigen activation), TGF-β induced inhibition of CAR-T cell function. Only CAR-T cells that secreted TGFβ regulators (e.g., TGFβR2 VHH dimers) were protected from the inhibitory effects of TGF-β (1 ng/ml or 10 ng/ml) stimulation (Figures 18A-18C). The inhibitory effect on CAR-T killing was associated with inhibition of proliferation and induction of the exhaustion marker Lag3.

Example 14. Repeated Antigenic Stimulation in Mesothelin (Msln)-Positive Tumors

Approximately 100,000 iPSC-derived anti-Msln CAR-T cells (co-expressing anti-Msln CAR and TGFβ regulators (e.g., TGFβR2-VH or dnTGFbR2)) or anti-GFP control VH (Msln-control VH) were co-cultured with 40,000 MiaPaca-2 tumor cells overexpressing human Msln in duplicate in the presence or absence of TGF-β (R&D Systems, 10 ng/ml). TGFβR2-VH is secreted by CAR-T cells, while dnTGFbR2 binds to the CAR-T cell membrane. Half of the CAR-T cells in each well were transferred to a new tumor cell plate (with or without TGF-β10 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 phenotyped (Figure 22A).

The viability of tumor cells was assessed using CellTiterGlo (Promega) according to the manufacturer's protocol. Plates were analyzed using a Pherastar plate reader. The percentage of killing was estimated using the following formula:

Control wells contained only tumor cells but no effector (ie, CAR-T) cells. The percentage of cytotoxicity is shown in Figure 22B.

Sytox Red dye (Thermofisher, according to the manufacturer's protocol) was used to exclude dead cells for cell counting, and an equal volume of cell suspension was obtained using an HTS unit on a Fortessa flow cytometer (BD Biosciences). Live CAR-T cells were counted by gating live cells, single cells, and size. The results were extrapolated to obtain the number of cells per well.

It was observed that after several rounds of restimulation with target cells (simulating chronic antigen activation), TGF-β induced inhibition of CAR-T cell function (i.e., killing) and suppressed CAR-T cell proliferation. Only CAR-T cells expressing TGFβ modulators (e.g., secreting TGFβR2 VH dimers or expressing membrane-bound dnTGFbR2) were protected from the inhibitory effects of 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

Equivalent programs 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 present invention is not intended to be limited by the above description, but rather is set forth in the appended claims.

Having described several aspects of at least one embodiment of the present invention, it will be appreciated that various changes, modifications and improvements will be apparent to those skilled in the art. Such changes, modifications and improvements are intended to be a part of this disclosure and are intended to fall within the spirit and scope of the present invention. Therefore, the foregoing description and drawings are intended only as examples, and the present invention is described in detail by the appended claims.

The use of ordinal terms such as "first", "second", "third", etc. in the claims to modify the claim elements themselves does not mean any priority, precedence or order of one claim element relative to another claim element, or the time order of method performance, but is merely an indicator to distinguish one claim element with a specific name from another element with the same name (but using the ordinal terms) in order to distinguish the claim elements.

As used herein in the specification and claims, the articles "a" and "an" shall be understood to include plural references unless expressly indicated to the contrary. Unless otherwise indicated, a claim or description including "or" between one or more group members is deemed to be satisfied if one, more than one, or all members of the group are present in, used in, or otherwise related to a given product or process, unless otherwise indicated by the context to the contrary or otherwise clearly indicated. The present invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The present invention also includes embodiments in which more than one or the entire group members are present in, used in, or otherwise related to a given product or process. In addition, it should be understood that the present invention covers 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 related thereto) in another dependent claim, unless otherwise indicated or unless a contradiction or inconsistency is obvious to a person of ordinary skill in the art. Where elements are presented in a list form (e.g., in Markush groups or similar format), it is understood that each subgroup of the elements is also disclosed and that any element may be removed from the group. It is understood that, in general, where the invention or aspects of the invention are referred to as comprising specific elements, features, etc., certain embodiments of the invention or aspects of the invention consist of or are substantially composed of such elements, features, etc. For simplicity, these embodiments are not specifically described in so many words in each case herein. It is also understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is stated in the specification. Publications, websites, and other reference materials cited to describe the background of the invention and to provide additional details about its implementation are hereby incorporated by reference.

Claims (20)

1. A genetically engineered T cell population comprising a chimeric antigen receptor (CAR) that recognizes cancer-related antigens and a TGFβ signaling pathway modulator. 2. The cell population of claim 1, wherein the CAR recognizes an antigen selected from the group consisting of ADGRE2, CLEC12, CAIX, CEA, CD5, 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 18.2, EGP-2, EGP-40, EpCAM, erb-B2, 3,4. FBP, fetal acetylcholine receptor, folate receptor-a, GCC (also known as GUCY2C), 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 embryonic antigen (h5T4), PSCA, PSMA, PTK7, ROR1, TAG-72, TROP2, VEGF -R2 and WT-1. 3. The cell population of claim 1 or 2, wherein the TGFβ signaling pathway modulator binds to TGFβ or a TGFβ receptor. 4. The cell population according to any one of the preceding claims, wherein the TGFβ signaling pathway modulator comprises an amino acid sequence selected from Table 1. 5. The cell population according to any one of the preceding claims, wherein the CAR is a CD19 CAR or a GCC CAR. 6. The cell population of claim 1, wherein the cells are autologous. 7. The cell population of claim 1, wherein the cells are allogeneic. 8. The cell population of any one 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 modulator of the TGFβ signaling pathway. 9. The cell population of any one 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. nucleic acids. 10. The cell population of any one of the preceding claims, wherein 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 domains, or combinations and variations thereof. 11. The cell population of any one of the preceding claims, wherein 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 combination 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 immune cell population according to claim 1. 18. A method of modulating an immune response in a host, the method comprising administering to the host a population of cells according to claim 1, wherein the modulation of the immune response comprises one of the following by host immune cells or More: increased IFNγ production; increased IL-2 production; increased antigen presentation; and increased proliferation. 19. A method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject an effective amount of the cell population of claim 1. 20. The method of claim 19, wherein the cancer is selected from the group consisting of: leukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic Leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin lymphoma tumour, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, vascular Sarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovialoma, 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 carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma, bile duct Carcinoma, 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, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma , neuroblastoma, retinoblastoma and their metastasis.