GCC-targeting CAR and multispecific binding molecules

Abstract

The compositions and methods described herein relate to the treatment of solid tumors using CAR T therapy. For example, the composition comprises a CAR T cell comprising an extracellular domain that binds to OR2I1P, LY6G6D, LRRC15, LY6K, GCC, GFRA4, F2RL2, QRFPR, IQGAP3, SIGLEC15, HAVCR1, PSG9, KISS1R, PRAME, HCN4, DPEP3, TMEM270, HER2, SLC7A3, SPRR2F, SLC45A2, CHRM1, CHRNA2, STEAP1B, FCRL2, a luteinizing hormone receptor, EDB, or CLDN18.2.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and more specifically, relates to a CAR targeting GCC and a multi-specific binding molecule. Background Art

[0002] Most existing cancer treatment options include surgery, radiotherapy and chemotherapy, targeted therapy, and immunotherapy. The disadvantages of existing options include poor treatment efficacy for patients with advanced disease, adverse side effects, and poor quality of life for patients. Therefore, there is a need to develop improved cancer treatments. Summary of the invention

[0003] Embodiments of the present disclosure relate to such a discovery that some antigens have relatively low expression on tumor cells compared to their expression on normal tissues. In addition, although expressed in normal tissues, these antigens are specifically expressed in specific tissues (e.g., a group of cells or organs), and killing normal cells of tissues may not cause life-threatening events (e.g., complications) to the subject. Examples of non-essential tissues include organs such as prostate, breast or melanocytes. Therefore, embodiments of the present disclosure relate to chimeric antigen receptors (CARs) comprising an extracellular domain binding to at least one of these antigens and using cells comprising CAR to treat cancer.

[0004] Embodiments relate to compositions and methods for treating cancer using CAR cells. Embodiments relate to isolated nucleic acid sequences encoding CAR, wherein CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain of CAR binds to an antigen of a solid tumor. Antigens may include GCC.

[0005] [1. CAR comprising SEQ ID NO: 127 (1D2C5) or 125 (4A8F7)]

[0006] A first aspect of the present invention provides a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain, and wherein: the extracellular domain binds to guanylate cyclase C (GCC) and comprises the amino acid sequence SEQID NO: 127 or 125.

[0007] According to the CAR of any scheme of the first aspect, preferably, the extracellular domain binds to GCC, and the CAR comprises the amino acid sequence of SEQ ID NO: 132 or 133.

[0008] According to the CAR of any scheme of the first aspect, preferably, the intracellular domain comprises a CD3zeta signaling domain and one or more co-stimulatory signaling regions, and the co-stimulatory signaling region comprises an intracellular domain of a co-stimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

[0009] An isolated nucleic acid encoding the CAR described in any one of the schemes of the first aspect.

[0010] A vector comprising the aforementioned isolated nucleic acid.

[0011] A modified cell comprising the aforementioned isolated nucleic acid.

[0012] Preferably, the modified cell further comprises one or more of the following:

[0013] A nucleic acid encoding a dominant negative form of an inhibitory immune checkpoint molecule or its receptor, and the immune checkpoint molecule comprises programmed death receptor 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T lymphocyte attenuator (BTLA), T cell immunoglobulin mucin 3 (TIM-3), lymphocyte activation protein 3 (LAG-3), T cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4) or CD160;

[0014] Endogenous TRAC gene expression is reduced;

[0015] A nucleic acid encoding Htert or a nucleic acid encoding SV40LT, or a combination thereof;

[0016] a nucleic acid encoding a suicide gene;

[0017] Nucleic acids encoding IL-6, IFNγ, IL-12 and / or IL-2.

[0018] A composition comprising a cell population containing the aforementioned isolated nucleic acid or vector, or a composition comprising the aforementioned modified cell population.

[0019] Preferably, the cell population comprises lymphocytes, and the lymphocytes comprise T cells, NK cells, macrophages or dendritic cells.

[0020] Preferably, the lymphocytes are T cells, and at least a portion of the T cells comprise a CAR that binds to a cell surface molecule of a white blood cell (WBC).

[0021] Preferably, the WBCs are B cells;

[0022] The cell surface molecule of WBC is CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13; or

[0023] The cell surface molecules of WBC are CD19, CD20, CD22 or BCMA.

[0024] Preferably, wherein the composition comprises a pharmaceutical composition.

[0025] A multispecific binding molecule (PBM), wherein the PBM comprises at least one first binding domain that binds to a T cell and at least one second binding domain, and wherein the second binding domain comprises the amino acid sequence of SEQ ID NO: 127 or 125.

[0026] Preferably, the first binding domain comprises a scFv that binds CD3.

[0027] Preferably, wherein the PBM is a bispecific antibody.

[0028] [2. Humanized Antibody (4A8F7)]

[0029] The second aspect of the present invention provides a humanized antibody that binds to human guanylate cyclase C (GCC), wherein the humanized antibody comprises:

[0030] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148 or 150; and

[0031] A light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152 or 154.

[0032] Preferably, the humanized antibody comprises:

[0033] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152; or

[0034] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 154; or

[0035] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 150; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152; or

[0036] A heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 150; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 154.

[0037] An isolated nucleic acid encoding the aforementioned humanized antibody.

[0038] Preferably, the isolated nucleic acid comprises:

[0039] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 149; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 153; or

[0040] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 149; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 155; or

[0041] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 151; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 153; or

[0042] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 151; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 155.

[0043] [3. Humanized antibody (1D2C5)]

[0044] The third aspect of the present invention provides a humanized antibody that binds to human guanylate cyclase C (GCC), wherein the humanized antibody comprises:

[0045] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156, 158 or 160; and

[0046] A light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO:162 or SEQ ID NO:164.

[0047] Preferably, the humanized antibody comprises:

[0048] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162; or

[0049] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 164; or

[0050] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162; or

[0051] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 164; or

[0052] A heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 160; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162.

[0053] An isolated nucleic acid encoding the aforementioned humanized antibody.

[0054] Preferably, the isolated nucleic acid comprises:

[0055] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 157; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 163; or

[0056] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 157; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 165; or

[0057] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 159; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 163; or

[0058] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 159; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 165; or

[0059] A nucleic acid sequence encoding a heavy chain variable region (HVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 161; and a nucleic acid sequence encoding a light chain variable region (LVR) sequence comprising the nucleic acid sequence of SEQ ID NO: 163.

[0060] A composition comprising the humanized antibody according to any one of the second aspect and the third aspect and a pharmaceutically acceptable carrier.

[0061] A vector comprising the isolated nucleic acid according to any one of the second and third aspects.

[0062] A modified cell comprising the nucleic acid described in any one of the second and third aspects, or comprising the vector described in any one of the second and third aspects.

[0063] A drug for treating GCC-positive tumors, comprising the humanized antibody described in any one of the second and third aspects.

[0064] A drug for treating GCC-positive related disorders, comprising the humanized antibody according to any one of the second and third aspects.

[0065] [IV. CAR formed based on the heavy and light chain sequences of humanized antibody (4A8F7)]

[0066] A fourth aspect of the present invention provides a chimeric antigen receptor (CAR), wherein the CAR comprises a scFv;

[0067] The scFv comprises a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148 or 150; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152 or 154.

[0068] Preferably, the scFv comprises:

[0069] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152; or

[0070] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 148; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 154; or

[0071] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 150; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 152; or

[0072] A heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 150; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 154.

[0073] [V. CAR formed based on the heavy and light chain sequences of humanized antibody (1D2C5)]

[0074] A fifth aspect of the present invention provides a chimeric antigen receptor (CAR), wherein the CAR comprises a scFv;

[0075] The scFv comprises a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156, 158 or 160; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 164.

[0076] Preferably, the scFv comprises:

[0077] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162; or

[0078] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 156; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 164; or

[0079] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162; or

[0080] a heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 164; or

[0081] A heavy chain variable region (HVR) sequence comprising the amino acid sequence of SEQ ID NO: 160; and a light chain variable region (LVR) sequence comprising the amino acid sequence of SEQ ID NO: 162.

[0082] [Six. Nucleic Acids, Vectors, Cells, and Compositions Encoding the CAR of the Fourth Aspect or the Fifth Aspect]

[0083] The sixth aspect of the present invention provides an isolated nucleic acid encoding the chimeric antigen receptor (CAR) described in any one of the fourth aspect or the fifth aspect.

[0084] A vector comprising the isolated nucleic acid described in the sixth aspect.

[0085] A modified cell comprising the vector described in the sixth aspect.

[0086] A composition comprising the chimeric antigen receptor (CAR) described in any one of the fourth aspect or the fifth aspect, or comprising the isolated nucleic acid described in the sixth aspect, or comprising the vector described in the sixth aspect, or comprising the modified cell described in the sixth aspect, and a pharmaceutically acceptable carrier.

[0087] A drug for treating GCC-positive tumors, comprising the CAR described in any one of the fourth aspect or the fifth aspect.

[0088] A drug for treating a GCC-positive related disorder, comprising the CAR described in any one of the fourth aspect or the fifth aspect.

[0089] This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The detailed description is made with reference to the accompanying drawings. The use of the same reference numerals in different drawings indicates similar or identical items.

[0091] Figure 1 Shows System construction and GCC expression pattern, where: 1A shows the IHC staining results of GCC antigen in normal tissues and cancer tissues. The stained tissues are (A1) normal esophageal squamous epithelium, (A2) normal gastric mucosa, (A3) normal small intestine tissue, and (A4) colorectal cancer tissue, indicating that the antigen GCC is a potential target for CAR-T therapy of colorectal cancer;

[0092] 1B shows Construction of the system, which consists of two components, GUCY2CCAR and CD19CAR;

[0093] 1C shows The expression of the two components of the system, GUCY2CCAR and CD19CAR.

[0094] Figure 2 The expression of GCC antigen in various cancer tissues is shown.

[0095] Figure 3A and 3B Flow cytometry results showing CAR expression on T cells.

[0096] Figure 4A and 4B Shown are the flow cytometry results of activated CAR T cells after co-culture with the corresponding substrates using CD8 and CD137 as markers.

[0097] Figure 5A and 5B Shown are the flow cytometry results of activated CART cells after co-culture with corresponding substrate cells using CD4 and CD40L as markers.

[0098] Figure 6 Cytokine release of CAR T cells after co-culture with corresponding substrate cells is shown.

[0099] Figure 7 Schematic diagram showing exemplary vectors and modified cells.

[0100] Figure 8 A schematic diagram showing an exemplary structure of a modification unit and an implementation of the modification unit is shown.

[0101] Fig. 9 The anti-tumor activity of 6708 and 6709CAR T cells was shown.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For purposes of the present disclosure, the following terms are defined as follows.

[0103] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0104] By “about” we mean an amount, level, value, quantity, frequency, percentage, dimension, size, number, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 4, 3, 2 or 1% from the referenced amount, level, value, quantity, frequency, percentage, dimension, size, number, weight or length.

[0105] As used herein, the term "activated" refers to a state of cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" particularly refers to T cells that are undergoing cell division.

[0106] The term "antibody" is used in the broadest sense to refer to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity or function. The antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, and Fv, Fab, Fab' and F(ab')2 and fragments thereof, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0107] The term "antibody fragment" refers to a portion of a full-length antibody, such as the antigen-binding region or variable region of an antibody. Other examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0108] The term "Fv" refers to the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in tight, non-covalent association. The folding of these two domains produces six hypervariable loops (3 loops each from the heavy and light chains) that donate the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv that includes only the three complementarity determining regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site (dimer).

[0109] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformations. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two major antibody light chain isotypes.

[0110] The term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage. The term also includes antibodies produced by synthesizing a DNA molecule encoding the antibody and expressing the DNA molecule to obtain the antibody or to obtain amino acids encoding the antibody. Synthetic DNA is obtained using techniques known in the art.

[0111] In an embodiment, the antibody is a heavy chain single variable domain (VHH) antibody, also known as a nanobody, discovered about 25 years ago. Heavy chain antibodies (HcAbs) are naturally produced by camelids and sharks. The antigen binding portion of HcAbs consists of VHH fragments.

[0112] The term "antigen" refers to a molecule that stimulates an immune response, which may involve antibody production or activation of specific immunocompetent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA includes a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that triggers an immune response, and thus encodes the term "antigen" as used herein. Antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. Antigens can be produced, synthesized or derived from biological samples including tissue samples, tumor samples, cells or biological fluids.

[0113] As used herein, the term "anti-tumor effect" refers to an effect associated with a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, a reduction in tumor cell proliferation, tumor cell survival, an increase in the life expectancy of a subject with tumor cells, or an improvement in various physiological symptoms associated with cancer. An "anti-tumor effect" may also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis in the first place.

[0114] The term "self-antigen" or "autoantigen" refers to an antigen that is mistakenly recognized as foreign by the immune system. Self-antigens include cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0115] The term "autologous" is used to describe material derived from a subject that is subsequently reintroduced into the same subject.

[0116] The term "allogeneic" is used to describe a transplant that originates from a different subject of the same species. For example, the donor subject may or may not be related to the recipient subject, but the donor subject has similar immune system markers to the recipient subject.

[0117] The term "xenogeneic" is used to describe a transplant that originates from a subject of a different species. For example, the donor subject is from a different species than the recipient subject, and the donor subject and recipient subject may not be genetically and immunologically compatible.

[0118] The term “cancer” is used to refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0119] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancers treated with the CAR disclosed herein include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0120] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (inactive forms and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0121] Solid tumors are abnormal tissue masses that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Chorionic carcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and central nervous system tumors (such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also called glioblastoma multiforme), astrocytomas, central nervous system lymphomas, germ cell tumors, medulloblastomas, cranial nerve sheath tumors, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0122] Solid tumor antigens are antigens expressed on solid tumors. In embodiments, solid tumor antigens are also expressed at low levels on healthy tissues. Examples of solid tumor antigens and their associated disease tumors are provided in Table 1.

[0123] Table 1

[0124]

[0125]

[0126] Throughout this specification, unless the context requires otherwise, the words "comprise", "include" and "comprising" will be understood to imply the inclusion of stated steps or elements (ingredients or components) or groups of steps or elements (ingredients or components) but not the exclusion of any other steps or elements or groups of steps or elements.

[0127] The phrase “consisting of…” is intended to include and be limited to whatever follows the phrase “consisting of…” Thus, the phrase “consisting of…” indicates that the listed elements or steps are required or mandatory, and no other elements may be present.

[0128] The phrase "consisting essentially of" is intended to include any elements listed after the phrase, and may include other elements or steps that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements or steps. Thus, the phrase "consisting essentially of" indicates that the listed elements or steps are required or mandatory, but other elements or steps are optional and may or may not be present, depending on whether they affect the listed elements or actions. In embodiments, those elements or steps that do not affect the embodiment are those that do not alter in a statistically significant manner the ability of the embodiment to perform a function in vitro or in vivo (e.g., kill cancer cells in vitro or in vivo).

[0129] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA". Complementarity can be "partial", in which only the bases of some nucleic acids match according to the base pairing rules, or there can be "complete" or "perfect" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0130] The term "corresponding to" or "corresponding to" refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or that encodes an amino acid sequence that is identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to an amino acid sequence in a reference peptide or protein.

[0131] The term "co-stimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing the signal to mediate T cell responses, including at least one response in proliferation, activation, differentiation, and other cellular responses, in addition to the primary signal provided by, for example, the binding of a TCR / CD3 complex to an MHC molecule loaded with a peptide. Co-stimulatory ligands may include B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, ligands of CD7, agonists or antibodies that bind to a Toll ligand receptor, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include agonists or antibodies that specifically bind to co-stimulatory molecules present on T cells, such as CD27, CD28, 4-1BB, 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.

[0132] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as proliferation. Costimulatory molecules include MHC class I molecules, BTLA and Toll-like receptors.

[0133] The term "co-stimulatory signal" refers to a signal that, in combination with a primary signal (eg, TCR / CD3 ligation), results in T cell proliferation and / or up- or down-regulation of key molecules.

[0134] The terms "costimulatory signaling region," "costimulatory domain," and "costimulatory domain" are used interchangeably to refer to one or more additional stimulatory domains in addition to a stimulatory or signaling domain (e.g., CD3 zeta). The terms "stimulatory" or "signaling" domains (or regions) are also used interchangeably, e.g., when referring to CD3 zeta, the primary signaling domain. In embodiments, the costimulatory signaling domain and the stimulatory signaling domain, e.g., the primary signaling domain, can be on the same molecule or on different molecules in the same cell.

[0135] The words "disease" and "disorder" may be used interchangeably or may differ in that a particular disease or disorder may have no known pathogen (and therefore a cause not yet determined) and therefore not yet recognized as a disease, but only as an adverse condition or syndrome for which clinicians have identified a set of more or less specific symptoms. The term "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease does not improve. In contrast, a "disorder" in a subject is a state of health in which the animal is able to maintain homeostasis, but the animal is not as healthy as it would be in the absence of the disease. A disease does not necessarily lead to a further decline in the animal's health if left untreated.

[0136] The term "effective" means sufficient to achieve a desired, expected, or intended result. For example, an "effective amount" in the context of treatment can be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

[0137] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have any of the following characteristics: a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand, whose nucleotide sequence is identical to the mRNA sequence (except that "T" is replaced by "U") and is generally provided in a sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of the gene or cDNA.

[0138] The term "exogenous" refers to a molecule that is not naturally present in a wild-type cell or organism but is usually introduced into a cell by molecular biology techniques. Examples of exogenous polynucleotides include vectors, plasmids and / or artificial nucleic acid constructs encoding the desired protein. With respect to polynucleotides and proteins, the terms "endogenous" or "natural" refer to naturally occurring polynucleotides or amino acid sequences that can be found in a given wild-type cell or organism. Moreover, a specific polynucleotide sequence that is separated from a first organism and transferred to a second organism by molecular biology techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence relative to the second organism. In a specific embodiment, a polynucleotide sequence can be "introduced" into a microorganism that already contains such a polynucleotide sequence by molecular biology techniques, for example, to produce one or more additional copies of the originally naturally occurring polynucleotide sequence, thereby promoting its overexpression. Encoded polypeptide.

[0139] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0140] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control (regulatory) sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked plasmids or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (AAV)) incorporating recombinant polynucleotides.

[0141] The term "homologous" refers to when two positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if two polypeptide DNA molecules are occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Comparison is when two sequences are aligned to give maximum homology.

[0142] The term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced by most subjects in the primary immune response. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody reactions, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by releasing mediators from mast cells and basophils upon exposure to allergens.

[0143] The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany its natural state. The material can be a cell or a macromolecule, such as a protein or nucleic acid. For example, as used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from flanking sequences in its naturally occurring state, such as a DNA fragment that has been removed from what would normally be the normal sequence and adjacent to the fragment. Alternatively, as used herein, an "isolated peptide" or "isolated polypeptide" etc. refers to the in vitro separation and / or purification of a peptide or polypeptide molecule from its natural cellular environment, as well as from other components of the cell.

[0144] The term "substantially purified" refers to a material that is substantially free of components with which it is normally associated in its natural state. For example, a substantially purified cell refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring or natural state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their natural state. In embodiments, the cells are cultured in vitro. In embodiments, the cells are not cultured in vitro.

[0145] In the context of this disclosure, the following commonly occurring abbreviations for nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0146] Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain (one or more) introns in certain versions.

[0147] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, and therefore they are one of the most efficient methods of gene delivery vectors. Furthermore, the use of lentiviruses enables the integration of the genetic information into the host chromosome, resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0148] The term "modulate" refers to a detectable increase or decrease in the level of a response in a subject as compared to the level of the response in the subject in the absence of the treatment or compound, and / or the response in an otherwise identical but untreated subject. The term includes perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0149] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0150] The term "under transcriptional control" refers to a promoter that is operably linked to a polynucleotide and in the correct location and orientation to control transcription initiation by RNA polymerase and expression of the polynucleotide.

[0151] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to the abnormal expression level of a tumor antigen in cells from a disease area, such as a solid tumor within a particular tissue or organ associated with a patient to the expression level in normal cells from that tissue or organ. Patients characterized by solid tumors or hematological malignancies with overexpression of a tumor antigen can be determined by standard assays known in the art.

[0152] The term "parenteral administration" of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intrasternal injection or infusion techniques.

[0153] The terms "patient", "subject", and "individual" and the like are used interchangeably herein and refer to any animal, e.g., a mammal, e.g., a human, or any living organism amenable to the methods described herein. In embodiments, the patient, subject, or individual is a human or a mammal. In embodiments, the term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans and animals, e.g., dogs, cats, mice, rats, and transgenic species thereof.

[0154] A subject in need of treatment or in need of treatment includes a subject suffering from a disease, disorder or condition in need of treatment. A subject in need thereof also includes a subject in need of treatment to prevent a disease, disorder or condition. Thus, a subject may also be in need of prevention of a disease condition or condition. In an embodiment, the disease is cancer.

[0155] The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of either nucleotide. The term includes all forms of nucleic acids, including single-stranded and double-stranded forms.

[0156] The terms "polynucleotide variants" and "variants" and the like refer to polynucleotides that show substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize to a reference sequence under stringent conditions defined below. These terms also include polynucleotides that differ from a reference polynucleotide by adding, deleting or replacing at least one nucleotide. Therefore, the terms "polynucleotide variants" and "variants" include polynucleotides in which one or more nucleotides have been added or deleted or replaced by different nucleotides. In this regard, it is well known in the art that certain changes including mutations, additions, deletions and substitutions can be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has a relationship with the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and between at least 51% and at least 99% and all integer percentages, such as 90%, 95% or 98%) sequence identity with a reference polynucleotide sequence described herein. The terms "polynucleotide variants" and "variants" also include naturally occurring allelic variants and orthologs.

[0157] The terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. In embodiments, polypeptides may include enzymatic polypeptides or "enzymes," which generally catalyze (i.e., increase the rate of) various chemical reactions.

[0158] The term "polypeptide variant" refers to a polypeptide that is distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue. In an embodiment, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In an embodiment, a polypeptide variant comprises a conservative substitution, and in this regard, it is well known in the art that certain amino acids can be changed to other amino acids with roughly similar properties without changing the nature of the polypeptide activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced by different amino acid residues.

[0159] The term "promoter" refers to a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0160] "NFAT promoter" refers to one or more NFAT binding sites or motifs connected to the minimum promoter of any gene expressed by T cells. In an embodiment, the minimum promoter of a gene expressed by a T cell is a minimum human IL-12 promoter. NFAT (nuclear factor of activated T cells) is a transcription factor. Examples of NFAT transcription factors include NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. These transcription factors bind to the NFAT binding site or motif in the NFAT promoter. The NFAT promoter (or its functional part or functional variant) can include any number of binding motifs, such as at least two, at least three, at least four, at least five or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at most twelve binding patterns. In an embodiment, the NFAT promoter includes six NFAT binding motifs.

[0161] The NFAT promoter (or its functional part or functional variant) is operably associated with the nucleotide sequence encoding IL-12 (or its functional part or functional variant). "Operably associated" means that when the NFAT protein binds to the NFAT promoter sequence (or its functional part or functional variant), the nucleotide sequence encoding IL-12 (or its functional part or functional variant) is transcribed into IL-12mRNA. . Not bound by a particular theory, it is believed that NFAT is regulated by the calcium signaling pathway. In particular, it is believed that TCR stimulation (by, for example, antigen) and / or stimulation of the cell calcium signaling pathway (by, for example, PMA / ionomycin) increases intracellular calcium concentration and activates calcium channels. It is believed that the NFAT protein is subsequently dephosphorylated by calmodulin and translocated to the nucleus, where it binds to the NFAT promoter sequence (or its functional part or functional variant) and activates downstream gene expression. By providing a NFAT promoter (or a functional part or a functional variant thereof) operably associated with a nucleotide sequence encoding IL-12 (or a functional part or a functional variant thereof), the nucleic acids described herein advantageously make it possible to express IL-12 (or a functional part or a functional variant thereof) only when a host cell comprising the nucleic acid is stimulated by, for example, PMA / ionomycin and / or an antigen. More information can be found in U.S. Patent No.: 8,556,882, which is incorporated by reference.

[0162] The terms "bind", "bind to" or "interact with..." refer to molecules that recognize and adhere to a second molecule in a sample or organism but do not substantially recognize or adhere to other structurally unrelated molecules in the sample. The term "specific binding" as used herein with respect to antibodies refers to antibodies that recognize a specific antigen but do not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, this cross-species reactivity itself does not change the classification of the antibody to specificity. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the classification of the antibody to specificity. In some cases, the term "specific binding" or "specific binding" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance to indicate that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure rather than any protein. If the antibody is specific for epitope "A", then the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0163] A "binding protein" is a protein that is capable of non-covalently binding to another molecule. A binding protein can bind, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), and / or a protein molecule (protein binding protein). In the case of a protein binding protein, it can bind to itself (forming homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of one or more different proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding, and protein binding activities.

[0164] A "zinc finger DNA binding protein" (or binding domain) is a protein, or domain within a larger protein, that binds DNA in a sequence-specific manner via one or more zinc fingers, which are binding domains whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

[0165] The zinc finger binding domain can be "engineered" to bind to a predetermined nucleotide sequence, for example, by modifying (changing one or more amino acids) the recognition helix region of a naturally occurring zinc finger protein. In addition, the zinc finger binding domain can be fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN) that targets a specific desired DNA sequence. For example, a pair of ZFNs (e.g., ZFN-left arm and ZFN-right arm) can be engineered to target and cause modification of a specific desired DNA sequence (e.g., a TRAC gene).

[0166] "Cleaving" refers to the breakage of the covalent backbone of a DNA molecule. Cutting can be initiated by a variety of methods, including but not limited to enzymatic hydrolysis or chemical hydrolysis of phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage may occur as a result of two different single-strand cleavage events. DNA cleavage can result in the production of flat ends or staggered ends. In embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.

[0167] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient binding conditions exist. For example, the sequence 5'GAATTC 3' is a target site for the Eco RI restriction endonuclease.

[0168] A "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be molecules of the same chemical type or molecules of different chemical types. Examples of the first class of fusion molecules include, but are not limited to, fusion proteins (e.g., fusions between a ZFP DNA binding domain and one or more activation domains) and fusion nucleic acids (e.g., encoding the above fusion proteins). Examples of the second class of fusion molecules include, but are not limited to, fusions between triplex-forming nucleic acids and polypeptides, and fusions between minor groove adhesives and nucleic acids.

[0169] Expression of fusion proteins in cells can be produced by delivering the fusion protein to the cell or by delivering a polynucleotide encoding the fusion protein to the cell, wherein the polynucleotide is transcribed and the transcript is transcribed to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation can also be involved in the expression of proteins in cells. Methods for delivering polynucleotides and polypeptides to cells are described elsewhere in this disclosure.

[0170] "Regulation" of gene expression refers to changes in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cutting, altering, inactivating, random mutations) can be used to regulate expression. Gene inactivation refers to any reduction in gene expression compared to cells that do not contain the ZFPs described herein. Therefore, gene inactivation may be partial or complete.

[0171] "Target region" is any region of cellular chromatin, such as a gene or non-coding sequence within or near a gene to which an exogenous molecule is desired to be bound. Binding can be for the purpose of targeted DNA cutting and / or targeted recombination. For example, the target region can be present in a chromosome, an episome, an organelle genome (e.g., mitochondria, chloroplasts), or an infectious viral genome. The target region can be within the coding region of a gene, within a transcribed non-coding region, such as a leader sequence, a trailing sequence, or an intron, or within a non-transcribed region, upstream or downstream of a coding region. The region of interest can be as small as a single nucleotide pair, or up to 2,000 nucleotide pairs, or any integer pair.

[0172] "Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used important metrics include p-values, which are the frequency or probability of the observed event if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less. "Reduction" or "reduction amount" is usually a "statistically significant" or physiologically significant amount, and can include a reduction of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 times or more (e.g., 100, 500, 1000 times) (including all integers and decimal points between 1 and above 1, such as 1.5, 1.6, 1.7.1.8, etc., the amount or level described here).

[0173] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event (e.g., signal transduction through a TCR / CD3 complex). Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure. In terms of primary response, CD3zeta is not the only suitable primary signaling domain for CAR construction. For example, as early as 1993, both CD3zeta and FcRy were demonstrated to be functional primary signaling domains for CAR molecules. Eshhar et al., "Specific activation and targeting of cytotoxic lymphocytes by chimeric single chains consisting of an antibody binding domain and either the gamma or zeta subunits of immunoglobulins and T cell receptors" PNAS, 1993 Jan 15;90(2):720- Figure 4 shows that two CAR constructs in which one scFv was fused to "either the FcRγ chain or the CD3 complex chain" triggered T cell activation and target cells. Notably, CAR constructs containing only the major signaling domains CD3 zeta or FcR gamma are functional in the absence of a co-present co-stimulatory domain, as demonstrated by Eshhar et al. Other non-CD3 zeta-based CAR constructs have been developed over the years. For example, Wang et al. (“Chimeric antigen receptors (CARs) based on killer immunoglobulin-like receptors (KIRs) trigger potent cytotoxic activity in solid tumors,” Molecular Therapy, Vol. 22, Supplement 1, May 2014, p. S57) tested a CAR molecule in which the scFv was fused to “the transmembrane and cytoplasmic domains of killer immunoglobulin-like receptors (KIRs)”. Wang et al. reported that “KIR-based CAR targeting mesothelin (SS 1-KIR) triggers antigen-specific cytotoxic activity and cytokine production comparable to CD3-based CARs.” A second publication from the same group, Wang et al. (“Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors” Cancer Immunol Res. 2015 Jul;3(7):815-26) showed that a CAR molecule in which “the single-chain variable fragment for antigen recognition was fused to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR)” functioned both in vitro and in vivo “when DAP12, an immunotyrosine-based adaptor containing an activation motif, was introduced into human T cells.”

[0174] The term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. For example, a functional signaling domain derived from a stimulatory molecule is a zeta chain associated with a T cell receptor complex. A stimulatory molecule includes a domain responsible for signal transduction.

[0175] The term "stimulatory ligand" refers to a ligand (referred to herein as a "stimulatory molecule") that can specifically bind to a cognate binding partner when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), thereby mediating the primary response of the T cell, including activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well known in the art and include, among others, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0176] The term "therapeutic agent" refers to treatment and / or prevention. A therapeutic effect is achieved by suppressing, alleviating or eradicating a disease state or alleviating the symptoms of a disease state.

[0177] The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or another clinician. The term "therapeutically effective amount" includes an amount of the compound that, when administered, is sufficient to prevent the development of one or more symptoms or symptoms of the condition or disease being treated or to alleviate to some extent one or more symptoms or symptoms of the condition or disease. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0178] The term "treating a disease" refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0179] The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. Cells include the primary subject cell and its progeny.

[0180] The term "vector" refers to a polynucleotide that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. The cell can be an in vitro cell or an in vivo cell in a subject. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term also includes non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc. For example, lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. Lentivirus vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: SIV. Lentivirus vectors are produced by multiple attenuation of HIV virulence genes, for example, genes env, vif, vpr, vpu and nef are deleted to make the vector biologically safe.

[0181] In an embodiment, the polynucleotides encoding antigen binding molecules and / or therapeutic agents can be used to implement the technology described herein. The method or use includes: providing a viral particle (e.g., AAV, slow virus or their variants) comprising a vector genome, wherein the vector genome comprises a polynucleotide, wherein the polynucleotide is operably connected to an expression control element that transcribes the polynucleotide; a certain amount of viral particles are applied to the subject so that the polynucleotide is expressed in the subject. In an embodiment, the AAV preparation may include AAV vector particles, empty capsids and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. For more information on viral particle administration and preparation, see U.S. Patent No.: US9840719 and Milani et al., Sci.Transl.Med.11,eaav7325 (2019) 22May 2019, which are incorporated herein by reference. In an embodiment, the polynucleotides may be integrated into the genome of the modified cell and the offspring of the modified cell will also express the polynucleotides, thereby producing a stably transfected modified cell. In an embodiment, the modified cell expresses a polynucleotide encoding CAR, but the polynucleotide is not integrated into the genome of the modified cell, so that the modified cell expresses the polynucleotide of transient transfection in a limited time period (e.g., a few days), after which the polynucleotide is lost due to cell division or other factors. For example, the polynucleotide is present in a modified cell in a recombinant DNA construct, mRNA or a viral vector, and / or the polynucleotide is mRNA, which is not integrated into the genome of the modified cell.

[0182] Range: Throughout this disclosure, various aspects of the disclosure may be given in a range format. It should be understood that the description in range format is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the disclosure. Therefore, the description of a range should be considered to have all possible sub-ranges disclosed specifically and a single numerical value within that range. For example, a description of a range such as from 1 to 6 should be considered to have specific disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and a single numerical value within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This applies regardless of the width of the range.

[0183] T cell responses in a subject refer to cell-mediated immunity associated with helper, killer, regulatory, and other types of T cells. For example, T cell responses may include activities such as assisting other white blood cells in immune processes and recognizing and destroying virus-infected cells and tumor cells. T cell responses in a subject can be measured by various indicators, such as the number of virus-infected cells and / or tumor cells killed by T cells, the amount of cytokines released by T cells, for example, infected cells and / or tumor cells when co-cultured with viruses, the level of proliferation of T cells in a subject, phenotypic changes in T cells (e.g., changes in memory T cells), and the lifespan or lifespan of T cells in a subject.

[0184] In the embodiment, the killing efficacy of CAR T cells can be measured by co-culturing CAR T cells with antigen-positive cells to perform in vitro killing assays.CAR T cells can be considered to have killing effects, TNF-α, etc. on corresponding antigen-positive cells by showing a decrease in the number of corresponding antigen-positive cells co-cultured with CAR T cells and an increase in the release of cytokines such as IFN-γ, compared with control cells that do not express the corresponding antigen. In addition, the in vivo anti-tumor activity of CAR T cells can be tested. For example, a xenograft model can be established in immunodeficient mice using antigens described herein. In the past two decades, human cancer cells or tumor biopsies have been xenografted into immunodeficient rodents (xenograft models), constituting the main preclinical screening cancer treatment for the development of new drugs (Song et al., Cancer Res.PMC August 21, 2014, and Morton et al., Nature Protocols, 2, -247-250 (2007)). To evaluate the anti-tumor activity of CAR T cells in vivo, the anti-tumor activity of CAR T cells was assessed in immunodeficient mice bearing tumor xenografts, for example, reduction of mouse tumors and / or mouse blood cytokines such as IFN-γ, TNF-α, etc.

[0185] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., a cytoplasmic domain). In an embodiment, the domains in the CAR polypeptide are on the same polypeptide chain (e.g., comprising a chimeric fusion protein). In an embodiment, the domains of the CAR polypeptide are not on the same molecule, e.g., discontinuously, or on different polypeptide chains.

[0186] In an embodiment, the intracellular signaling domain may include a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as described herein. In an embodiment, the intracellular signaling domain includes a functional signaling domain derived from a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In an embodiment, the intracellular signaling domain also includes one or more functional signaling domains derived from at least one co-stimulatory molecule. The co-stimulatory signal region refers to a portion of a CAR, including the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules may include cell surface molecules for inducing an effective response of lymphocytes (response antigens).

[0187] Between the extracellular domain and the transmembrane domain of CAR, a spacer domain may be incorporated. As used herein, the term "spacer domain" generally refers to any oligonucleotide or polypeptide that functions to connect a transmembrane domain to an extracellular domain and / or a cytoplasmic domain in a polypeptide chain. The spacer domain may include up to 300 amino acids, 10 to 100 amino acids, or 25 to 50 amino acids.

[0188] The extracellular domain of the CAR can include a target for a specific tumor marker (e.g., a tumor antigen). Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T cell-mediated immune response. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. For example, when the antigen bound by CAR is CD19, the CAR is called CD19 CAR (19CAR, CD19CAR, CD19 CAR or CD19-CAR), which is a CAR molecule comprising an antigen binding domain that binds to CD19.

[0189] In an embodiment, the extracellular ligand binding domain comprises an scFv comprising a light chain variable (VL) region and a heavy chain variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single-chain variable region fragments are made by connecting light chain and / or heavy chain variable regions using a short connecting peptide (Bird et al., Science 242: 423-426, 1988). An example of a connecting peptide is a GS linker having an amino acid sequence (GGGGS) 3 (SEQ ID: 2), which bridges about 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). Generally, the linker can be a short and flexible polypeptide comprising about 20 or fewer amino acid residues. The linker can be modified in turn to obtain additional functions, such as the attachment of a drug or attachment to a solid support. Single-chain variants can be produced by recombinant or synthetic means. For the synthetic production of scFv, an automatic synthesizer can be used. For the recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding scFv can be introduced into a suitable host cell, which can be a eukaryotic cell, such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell, such as Escherichia coli. The polynucleotide encoding the scFv of interest can be prepared by conventional operations such as the connection of the polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0190] In embodiments, tumor antigens include HER2, CD19, CD20, CD22, kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3 / 4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor α2, IL-11 receptor α, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-α, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, TEM1, TEM8 or virus-related antigens are expressed by tumors. In embodiments, the binding element of CAR includes any antigen binding portion that, when bound to its cognate antigen, affects tumor cells such that tumor cells cannot grow, are reduced in size or die.

[0191] CAR can be a bispecific CAR. For example, two antigen binding domains are located on the same CAR (bispecific CAR or tandem CAR (tanCAR)), different CAR molecules or CAR and T cell receptor (TCR). A single CAR can include two different antigen binding domains, or two different antigen binding domains are each located on a separate CAR. CAR can have more than two antigen binding domains, for example, multispecific CAR. The antigen binding domain of multispecific CAR can be on the same CAR or on different CARs, such as one antigen binding domain on each CAR.

[0192] In an embodiment, the intracellular domain of the CAR comprises a co-stimulatory signaling region, which comprises the intracellular domain of a co-stimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

[0193] In embodiments, the intracellular domain comprises a CD3 zeta signaling domain. Embodiments relate to vectors comprising an isolated nucleic acid sequence described herein. Embodiments relate to isolated cells comprising an isolated nucleic acid sequence described herein.

[0194] The cells described herein, including CAR cells and modified cells, can be derived from stem cells. Stem cells can be adult stem cells, embryonic stem cells or non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Cells can also be dendritic cells, NK cells, B cells or T cells, selected from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes and helper T lymphocytes. In an embodiment, the cell can be derived from a group consisting of CD4+T lymphocytes and CD8+T lymphocytes. Before the cells described herein are amplified and genetically modified, a cell source can be obtained from a subject by a variety of non-limiting methods. T cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue and tumors. In an embodiment, any number of T cell lines available and known to those skilled in the art can be used. In an embodiment, cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with infection. In embodiments, the cell is part of a mixed population of cells that exhibit different phenotypic characteristics.

[0195] A cell population refers to a group of 2 or more cells. The cells of the population can be the same, so that the population is a homogeneous cell population. The cells in the population may be different, so that the population is a mixed population or a heterogeneous population of cells. For example, a mixed cell population may include modified cells comprising a first CAR and cells comprising a second CAR, wherein the first CAR and the second CAR bind to different antigens. The term "stem cell" refers to any type of cell that has the ability to self-renew and differentiate into other types of cells. For example, a stem cell produces two daughter stem cells (occurring in vitro with the culture of embryonic stem cells) or a stem cell and a cell that undergoes differentiation (As occurs in hematopoietic stem cells, hematopoietic stem cells produce blood cells). Different types of stem cells can be distinguished based on their origin and / or their ability to differentiate into other types of cells. Stem cells can include any type of stem cell, such as embryonic stem cells (ES cells) (i.e., pluripotent stem cells), somatic stem cells, induced pluripotent stem cells, etc.

[0196] Pluripotent embryonic stem cells can be found in the inner cell mass of the blastocyst and have a high innate differentiation capacity. For example, pluripotent embryonic stem cells have the potential to form any type of cell in the body. When grown in vitro for a long time, ES cells maintain pluripotency, and daughter cells retain the potential for multidirectional differentiation.

[0197] Somatic stem cells can include fetal stem cells (derived from a fetus) and adult stem cells (found in various tissues, such as the bone marrow). These cells are thought to have a lower differentiation capacity than pluripotent embryonic stem cells - fetal stem cells have a higher capacity than adult stem cells; they apparently only differentiate into a limited number of different cell types and are described as pluripotent cells. "Tissue-specific" stem cells typically produce only one type of cell. For example, embryonic stem cells can differentiate into blood stem cells (e.g., hematopoietic stem cells (HSCs)), which can further differentiate into various blood cells (e.g., red blood cells, platelets, white blood cells, etc.).

[0198] Induced pluripotent stem cells (iPS cells or iPSCs) can include a class of pluripotent stem cells artificially derived from non-pluripotent cells (e.g., adult cells) by inducing the expression of specific genes. Induced pluripotent stem cells are similar to natural pluripotent stem cells in many ways, such as embryonic stem cells (ES), such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid formation, teratoma formation, viable chimera formation, and efficacy and distinguishability. Induced pluripotent cells can be isolated from adult stomach, liver, skin cells, and blood cells.

[0199] In embodiments, the CAR cell, modified cell or cell is a T cell, NK cell, macrophage or dendritic cell. For example, the CAR cell, modified cell or cell is a T cell.

[0200] T cells or T lymphocytes are a type of white blood cell of the immune system. There are several types of T cells, including T helper (TH) cells, cytotoxic T (TC) cells (T killer cells, killer T cells), natural killer T (NKT) cells, memory T (Tm) cells, regulatory T (Treg) cells, and gamma delta T (γδT) cells.

[0201] T helper (TH) cells assist other lymphocytes, for example, activating cytotoxic T cells and macrophages and maturing B cells into plasma cells and memory B cells. These T helper cells express the CD4 glycoprotein on their surface and are also called CD4+ T cells. Once activated, these T cells divide rapidly and secrete cytokines.

[0202] Cytotoxic T (TC) cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. They express the CD8 protein on their surface. Cytotoxic T cells release cytokines.

[0203] Natural killer T (NKT) cells are different from natural killer cells. NKT cells recognize glycolipid antigens presented by CD1d. Once activated, NKT cells produce cytokines and release cell-killing molecules.

[0204] Memory T (Tm) cells have a long lifespan and can expand into a large number of effector T cells after re-exposure to their cognate antigens. Tm cells provide the immune system with memory of previously encountered pathogens. There are multiple subtypes of Tm cells, including central memory T (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TRM) cells, and virtual memory T cells. Tm cells are CD4+ or CD8+, usually CD45RO.

[0205] Regulatory T (Treg) cells shut down T cell-mediated immunity at the end of an immune response and suppress autoreactive T cells that escape the thymic negative selection process. Treg cell subsets include thymic Tregs and peripherally derived Tregs. Both subsets of Tregs require the expression of the transcription factor FOXP3.

[0206] γδT cells are a subset of T cells that have a γδT cell receptor (TCR) on the cell surface, as most T cells express αβTCR chains. γδT cells are rare in humans and mice, and are mainly found in the intestinal mucosa, skin, lungs, and uterus. They are involved in the initiation and propagation of immune responses.

[0207] In embodiments, the antigen binding molecule is a T cell receptor (TCR). In embodiments, TCR is a modified TCR. In embodiments, TCR is derived from tumor-specific T cells spontaneously generated in the patient. In embodiments, TCR binds to tumor antigens. In embodiments, tumor antigens include CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1. In embodiments, TCR includes TCRγ and TCRδ chains or TCRα and TCRβ chains.

[0208] In embodiments, T cell clones expressing TCRs with high affinity for target antigens can be isolated. In embodiments, tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) can be cultured in the presence of antigen presenting cells (APCs), which are pulsed with peptides representing epitopes, known to be presented in the context of defined HLA alleles. High affinity clones can then be selected based on the ability of MHC-peptide tetramer staining and / or recognition and lysis of target cells pulsed with low titration concentrations of homologous peptide antigens. After selecting clones, TCRα and TCRβ chains or TCRγ and TCRδ chains are identified and separated by molecular cloning. For example, for TCRα and TCRβ chains, expression constructs are then generated using TCRα and TCRβ gene sequences, ideally promoting stable, high-level expression of two TCR chains in human T cells. Transduction vectors (e.g., γ retroviruses or lentiviruses) can then be generated and tested for function (antigen specificity and functional affinity), and used to produce clinical batches of vectors. An aliquot of the final product is then used to transduce a target T cell population (usually purified from patient PBMCs), which is expanded prior to infusion into the subject.

[0209] In embodiments, APCs include dendritic cells, macrophages, Langerhans cells, and B cells or T cells.

[0210] In an embodiment, the binding element of a CAR can include any antigen binding portion that, when bound to its cognate antigen, affects tumor cells, e.g., it kills tumor cells, inhibits the growth of tumor cells, or promotes the death of tumor cells.

[0211] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to contain the same gene, or by isolation directly from cells and tissues containing the same using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0212] Embodiments of the present disclosure also relate to vectors into which nucleic acids described herein are inserted. Vectors can be derived from retroviruses, such as lentiviruses, which are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and propagation in daughter cells. Lentiviral vectors have more advantages than vectors derived from oncogenic retroviruses (such as murine leukemia viruses) because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0213] Viruses can be used to deliver nucleic acids to cells in vitro and in vivo (in a subject). Examples of viruses that can be used to deliver nucleic acids to cells include retroviruses, adenoviruses, herpes simplex viruses, vaccinia viruses, and adeno-associated viruses.

[0214] There are also non-viral methods for delivering nucleic acids into cells, such as electroporation, gene guns, sonoporation, magnetofection, and the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.

[0215] The expression of natural or synthetic nucleic acids encoding CAR is usually achieved by operably connecting the nucleic acid encoding the CAR polypeptide or part thereof to one or more promoters and integrating the construct into an expression vector. The vector can be suitable for replication and integration into eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0216] Additional information regarding the expression and gene transfer of synthetic nucleic acids encoding CARs into mammalian cells is provided in U.S. Pat. No. 8,906,682, which is incorporated by reference in its entirety.

[0217] The pharmaceutical compositions of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, although the appropriate dosage can be determined by clinical trials.

[0218] When "immunologically effective amount", "anti-tumor effective amount", "tumor suppression effective amount", "therapeutic amount" or "effective amount" is indicated, the exact amount of the composition of the present disclosure can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). It can be said that the pharmaceutical composition comprising the T cells described herein can be 10 4 Up to 10 9 cells / kg body weight, preferably 10 5 Up to 10 6The dosage of individual cells / kg body weight is administered, including all integer values ​​within those ranges. T cell compositions can also be administered multiple times at these doses. Cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by those skilled in the art by monitoring the patient's disease signs and adjusting the treatment accordingly. In an embodiment, activated T cells are administered to the subject, followed by a blood draw (or apheresis). T cells are collected, amplified and re-injected into the subject. This process can be performed several times every few weeks. In an embodiment, T cells can be activated from a blood draw of 10cc to 400cc. In an embodiment, T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc or 100cc. Without being bound by theory, certain T cell populations can be selected using this multiple blood draw / multiple re-infusion scheme.

[0219] The administration of pharmaceutical compositions described herein can be carried out in any convenient manner, including by aerosol inhalation, injection, intake, blood transfusion, implantation or transplantation. Pharmaceutical compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (iv) or intraperitoneally. In embodiments, T cell compositions of the present disclosure are administered to patients by intradermal or subcutaneous injection. In an embodiment, T cell compositions of the present disclosure are administered by intravenous injection. The composition of T cells can be directly injected into tumors, lymph nodes or infection sites. In embodiments of the present disclosure, cells activated and amplified using methods described herein or other methods known in the art wherein T cells are expanded to therapeutic levels are administered to patients with (for example, before, at the same time or afterwards) any number of related treatment modalities, including but not limited to antiviral therapy, cidofovir and interleukin 2, cytarabine (also referred to as ARA-C) or natalizumab treatment of MS patients or efalizumab treatment of psoriasis patients or other treatments such as drug therapy for PML patients. In further embodiments, the T cells of the present disclosure can be combined with chemotherapy, radiotherapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993; Isoniemi (supra)). In embodiments, the cell compositions of the present disclosure are administered with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In embodiments, the cell compositions of the present disclosure are administered after B cell ablation therapy, such as an agent reactive with CD20, such as Rituxan?. For example, a subject may receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the present disclosure. In embodiments, the expanded cells are administered before or after surgery.

[0220] The above therapeutic doses to be administered to a patient will vary with the exact nature of the condition being treated and the subject being treated. Depending on a variety of factors, physicians can scale human dosages according to practices recognized in the art.

[0221] Additional information regarding methods of cancer treatment using engineered or modified T cells is provided in U.S. Patent No. 5,315,418. U.S. Patent No. 8,906,682, incorporated by reference in its entirety.

[0222] In embodiments, the cell populations described herein are used for autologous CAR T cell therapy. In embodiments, the CAR T cell therapy is allogeneic CAR T cell therapy, TCR T cell therapy, and NK cell therapy.

[0223] Embodiments relate to an in vitro method for preparing modified cells. The method may include obtaining a cell sample from a subject. For example, the sample may include T cells or T cell progenitors. The method may further include transfecting cells with DNA encoding at least one CAR, and culturing a CAR cell population in vitro in a culture medium that selectively enhances the proliferation of T cells expressing CAR.

[0224] In embodiments, the sample is a cryopreserved sample. In embodiments, the cell sample is from umbilical cord blood or a peripheral blood sample from a subject. In embodiments, the cell sample is obtained by apheresis or venipuncture. In embodiments, the cell sample is a T cell subset.

[0225] As used herein, the term "gene fusion" refers to the fusion of at least a portion of a gene with at least a portion of another gene. Gene fusion does not need to include the entire gene or the exons of a gene. In some cases, gene fusion is associated with changes in cancer. Gene fusion products refer to chimeric genomic DNA, chimeric messenger RNA, truncated proteins or chimeric proteins produced by gene fusion. Gene fusion products can be detected by the various methods described in U.S. Patent 9,938,582, which is incorporated herein by reference. "Gene fusion antigen" refers to a truncated protein or chimeric protein produced by gene fusion. In embodiments, the epitope of a gene fusion antigen may include a portion of a gene fusion antigen or an immunogenic portion of another antigen caused by gene fusion. In embodiments, the gene fusion antigen interacts with a cell membrane, or is a portion of a cell membrane.

[0226] In embodiments, experimental methods such as qPCR and FACS can be used to detect mRNA and protein expression levels of target molecules (e.g., CAR and cytokines) in human cells. In addition, target molecules that are specifically expressed in corresponding tumor cells and expressed very low or undetectable in normal tissue cells can be identified.

[0227] In the embodiments, in vitro killing tests and killing experiments of CAR T cells co-cultured with antigen-positive cells can be performed. CAR T cells can show killing effects on corresponding antigen-positive cells, reduce the number of corresponding antigen-positive cells co-cultured with CAR T cells, increase the release of IFN-γ, TNF-α, etc. compared with control cells that do not express the corresponding antigen.

[0228] In the embodiments, an in vivo killing test can be performed. For example, tumor cells with corresponding antigens can be transplanted into mice to induce tumors, and CAR T cells can be infused to reduce tumors in mice, and the blood IFN-γ, TNF-α and other signaling defects in mice can be detected.

[0229] Embodiments relate to methods for inducing and / or enhancing T cell responses in subjects with solid tumors or treating solid tumors in subjects, the method comprising administering an effective amount of T cells comprising CAR as described herein. In embodiments, the intracellular domain of CAR includes a costimulatory signaling region, which includes an intracellular domain of a costimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. In embodiments, the intracellular domain includes a CD3zeta signaling domain.

[0230] Embodiments relate to vectors comprising the isolated nucleic acid described herein.

[0231] Embodiments relate to isolated cells comprising isolated nucleic acid sequences described herein. Embodiments relate to compositions comprising a T cell population comprising a CAR described herein. Embodiments relate to CARs encoded by isolated nucleic acid sequences described herein. Embodiments relate to methods of initiating and / or enhancing T cell responses or treating tumors in subjects, the methods comprising: administering an effective amount of T cells comprising a CAR described herein.

[0232] In an embodiment, the CAR molecules described herein include one or more complementary determining regions (CDRs) to bind to an antigen of interest. CDR is a part of the variable domain of immunoglobulins and T cell receptors for binding to specific antigens. Each variable domain has three CDRs. Due to the presence of variable heavy domains and variable light domains, there are six CDRs for binding antigens. In addition, since antibodies have two heavy chains and two light chains, antibodies can have a total of twelve CDRs for binding antigens.

[0233] In embodiments, the modified cells described herein include CAR molecules containing at least two different antigen binding domains.CAR molecules can be bispecific CAR molecules.For example, two antigen binding domains can be on the same CAR molecule, on different CAR molecules, or on CAR molecules and T cell receptors (TCR).Single CAR may include at least two different antigen binding domains, or these two different antigen binding domains are each located on a separate CAR molecule.At least two different antigen binding domains can be on the same CAR molecule or different CAR molecules, but in the same modified cells.In addition, at least two different antigen binding domains can be on CAR molecules and T cell receptors in the same modified cell.In an embodiment, a bispecific CAR molecule may include a binding domain for binding to a WBC antigen (e.g., CD19) and a binding domain for binding to a solid tumor antigen.In an embodiment, a bispecific CAR molecule may include two binding domains for binding to two different solid tumor antigens.

[0234] In embodiments, at least two different antigen binding domains are on different CAR molecules, and the CAR molecules are expressed by different modified cells. In addition, one or more different antigen binding domains are located on CAR molecules and T cell receptors expressed by different modified cells.

[0235] The present disclosure describes an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein: the extracellular domain binds guanylate cyclase C (GCC) and comprises the amino acid sequence of SEQ ID NO: 127 or 125.

[0236] In an embodiment, the extracellular domain binds GCC and the CAR comprises the amino acid sequence of SEQ ID NO: 132 or 133.

[0237] Embodiments relate to multispecific binding molecules (PBMs), wherein the PBM comprises at least one first binding domain that binds T cells and at least one comprising SEQ ID NOs: 127 or 125. In embodiments, the first binding domain comprises a scFv that binds CD3. In embodiments, the PBM is a bispecific antibody.

[0238] Embodiments relate to nucleic acids encoding CAR separation, wherein CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain binds to an antigen of a tumor (e.g., a solid tumor). Embodiments relate to vectors comprising isolated nucleic acids as described herein. Embodiments relate to CARs encoded by isolated nucleic acids or vectors as described herein. Embodiments relate to modified cells comprising isolated nucleic acids, vectors or CARs as described herein, and optionally modified cells comprise modified T cells. Embodiments relate to modified cell groups comprising modified cells, and optionally, wherein the modified cell groups comprise modified T cells. Embodiments relate to compositions comprising isolated nucleic acids, CARs, vectors, modified cells or modified cell groups as described herein, and optionally, the compositions comprise modified T cell groups. Embodiments relate to methods of initiating and / or enhancing T cell responses or treating solid tumors in subjects with solid tumors, the methods comprising administering an effective amount of isolated nucleic acids, vectors, CARs, an effective amount of modified T cells or modified T cell groups as described herein, or an effective amount of compositions as described herein. In embodiments, compositions as described herein include pharmaceutical compositions. Embodiments relate to kits comprising the nucleic acids, vectors, CARs, modified cells, modified cell populations, compositions and / or pharmaceutical compositions described herein.

[0239] Embodiments relate to an isolated nucleic acid encoding a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain binds a gene fusion neoantigen, and optionally, wherein the gene fusion comprises at least a portion of a first gene to at least a portion of a second gene, and wherein optionally, the gene fusion neoantigen is associated with a tumor.

[0240] Embodiments relate to methods of eliciting and / or enhancing a T cell response in a subject with a solid tumor or treating a solid tumor in a subject, the method comprising administering an effective amount of T cells comprising an isolated nucleic acid. Embodiments relate to implementing the methods described in PCT Publication Nos. WO2020146743 and WO2020106843. The method of the system is incorporated herein by reference in its entirety, and the CAR comprises a ScFv described herein.

[0241] Embodiments relate to multispecific binding molecules (PBMs), wherein the PBM comprises at least a first binding domain that binds to a T cell and at least a second binding domain comprises an scFv or antibody as described herein. Embodiments relate to a method of treating cancer comprising administering an effective amount of a PBM, such as a bispecific antibody ( ), more information on PBM can be found in PCT Application No: PCT / US21 / 28429 of ICT, which is incorporated herein by reference in its entirety. Embodiments relate to isolated nucleic acids encoding multispecific binding molecules comprising scFv or antibodies described herein.

[0242] In an embodiment, the intracellular domain comprises a signaling domain and a co-stimulatory signaling domain, and optionally, the co-stimulatory signaling domain comprises CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof. In an embodiment, the signaling domain comprises a CD3zeta signaling domain.

[0243] In embodiments, the signaling domain and the co-stimulatory signaling domain are located on different molecules in the same cell.

[0244] In embodiments, the modified cells comprise or are T cells derived from a healthy donor or a subject suffering from cancer.

[0245] In embodiments, the modified cell further comprises a dominant negative form of a receptor or ligand associated with an immune checkpoint inhibitor, and optionally the modified cell is a modified T cell. In embodiments, the immune checkpoint inhibitor is selected from programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuation factor (BTLA), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte activation protein 3 (LAG-3), T cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4) and CD 160. In embodiments, the immune checkpoint inhibitor is a modified PD-1. In the embodiment, the modified PD-1 lacks a functional PD-1 intracellular domain for PD-1 signal transduction; interferes with the pathway between human T cell PD-1 of a human cell and PD-L1 of a certain cell; comprises or is a PD-1 extracellular domain or a PD-1 transmembrane domain, or a combination thereof; comprises a modified PD-1 intracellular domain, including substitutions or deletions compared to the wild-type PD-1 intracellular domain; or comprises or is a soluble receptor comprising a PD-1 extracellular domain that binds to PD-L1 of a specific cell. In the embodiment, the inhibitory effect of PD-L1 on cytokine production by a population of human T cells is less than the inhibitory effect of PD-L1 on cytokine production by human T cells that do not contain at least a portion of the nucleic acid sequence encoding the modified PD-1.

[0246] In the embodiment, the modified cell is a modified T cell that is engineered to express and secrete a therapeutic agent, and optionally wherein the therapeutic agent is a cytokine or a small protein. In the embodiment, the therapeutic agent is or comprises IFN-γ. In the embodiment, the therapeutic agent is or comprises at least one of IL-6, IFN-γ, IL-17, IL-12, and CCL19. In the embodiment, the therapeutic agent is or comprises IL-15 or IL-12, or a combination thereof. In the embodiment, the therapeutic agent is or comprises a recombinant or natural cytokine. In the embodiment, the therapeutic agent comprises an FC fusion protein bound to a small protein. In the embodiment, the cytokine is or comprises IL-12, IL-15, IL-6, or IFN-γ. In the embodiment, the therapeutic agent is regulated by Hif1a, NFAT, FOXP3, and / or NFkB. In the embodiment, the therapeutic agent is or comprises two or more polynucleotides encoding recombinant or naturally occurring cytokines, and the polynucleotides are connected by 2A or IRES components.

[0247] In an embodiment, the present disclosure describes a first targeting vector and a second targeting vector, wherein the first targeting vector comprises a nucleic acid encoding a CAR that binds a blood antigen and a therapeutic agent, and the second targeting vector comprises a nucleic acid encoding a CAR that binds a solid tumor antigen and a dominant negative form of an immune checkpoint molecule.

[0248] In an embodiment, the modified cell, optionally a modified T cell, comprises a first targeting vector comprising a nucleic acid encoding a CAR that binds CD19 and a therapeutic agent and a second targeting vector comprising a nucleic acid encoding a CAR that binds UPK2, ACPP, SIGLEC15 or KISS1R and a dominant negative form of PD-1.

[0249] In an embodiment, the modified cell, optionally a modified T cell, comprises a first targeting vector comprising a nucleic acid encoding a CAR that binds a blood antigen and a second targeting vector comprising a nucleic acid encoding a CAR that binds a solid tumor antigen.

[0250] In an embodiment, the modified cell, optionally a modified T cell, comprises a first targeting vector comprising a nucleic acid encoding a CAR that binds a B cell antigen and a second targeting vector comprising a nucleic acid encoding a CAR that binds a solid tumor antigen.

[0251] In embodiments, the modified cells, optionally modified T cells, comprise nucleic acids encoding human telomerase reverse transcriptase (hTERT), simian vacuolating virus 40 large T (SV40LT) antigen (Ag), or a combination thereof. In embodiments, the modified T cells are more proliferative than T cells without the nucleic acid sequence. In embodiments, the modified T cells retain the function of normal T cells / CAR T cells, such as cell therapy function.

[0252] In an embodiment, the T cell comprises a CAR and is cultured in the presence of an agent recognized by the extracellular domain of the CAR. In an embodiment, the modified cell, optionally a modified T cell, comprises a genomic integration of a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof, and optionally wherein the cell comprises constitutive expression of hTERT, SV40LT, or a combination thereof. In an embodiment, the expression of hTERT, SV40LT, or a combination thereof is regulated by an inducible expression system such as the rtTA-TRE system.

[0253] In an embodiment, the modified T cell comprises a nucleic acid sequence encoding a suicide gene, such as the HSV-TK system.

[0254] In embodiments, the modified cells, optionally modified T cells, have reduced graft-versus-host disease (GVHD) responses in bioincompatible human recipients compared to the GVHD responses of primary human T cells. In embodiments, the modified cells, optionally modified T cells, have reduced endogenous TRAC gene expression.

[0255] Embodiments relate to antibodies or antibody fragments that bind to GCC, wherein the CDRs of the light chain variable region (LVR) of the antibody or antibody fragment comprise the amino acid sequence of SEQ ID NOs: 110-112, 114-116 or 122-124, and wherein the CDRs of the heavy chain variable region (HVR) of the antibody or antibody fragment comprise the amino acid sequence of SEQ ID NOS: 106-108 or 118-120.

[0256] In embodiments, the antibody or antibody fragment binds to GCC and comprises an LVR comprising the amino acid sequence of SEQ ID NO: 109, 113 or 121 and an HVR comprising the amino acid sequence of SEQ ID NO: 105 or 117. In embodiments, the LVR comprises the amino acid sequence of SEQ ID NO: 109 or 113, and the HVR comprises the amino acid sequence of SEQ ID NO: 105. In embodiments, the LVR comprises the amino acid sequence of SEQ ID NO: 121, and the HVR comprises the amino acid sequence of SEQ ID NO: 117.

[0257] In an embodiment, the HVRs are linked to a human IgG constant region, and the human IgG is IgG1 or IgG3.

[0258] In an embodiment, the antibody is conjugated to a cytotoxic agent, and the cytotoxic agent is a radioisotope or a toxin.

[0259] In an embodiment, the antibody or antibody fragment comprises or is an scFv, wherein the scFv comprises an LVR connected to an HVR via a linker, the HVR comprises SEQ ID NO: 105 or 117, and the LVR comprises SEQ ID NO: 109, 113, or 121. and / or wherein the scFv comprises SEQ ID NO: 127, SEQ ID NO: 105, a linker sequence and SEQ ID NO: 109; SEQ ID NO: 117, a linker sequence, and SEQ ID NO: 121; or SEQ ID NO: 125.

[0260] In an embodiment, the antibody or antibody fragment comprises one or more amino acid sequences comprising SEQ ID NOS: 105-130.

[0261] In an embodiment, a bispecific antibody comprises an antibody or antibody fragment described herein and an antibody that binds T cells.

[0262] Embodiments relate to polynucleotides encoding antibodies or antibody fragments that bind GCC.

[0263] Embodiments relate to a chimeric antigen receptor (CAR) comprising an antigen binding domain, the antigen binding domain comprising an antibody or antibody fragment that binds to GCC. In an embodiment, CAR comprises at least one of the amino acid sequences SEQ ID NOS: 105-130, 132, and 133. In an embodiment, CAR comprises SEQ ID NOS: 132 or 133. The embodiment relates to a bispecific CAR comprising CAR binding to GCC described herein. In an embodiment, CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises an antigen binding domain. In an embodiment, the intracellular domain comprises a stimulatory signal domain and a co-stimulatory signal domain, which comprises one or more intracellular domains of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigens -1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In an embodiment, the stimulatory signaling domain of CAR comprises a CD3 zeta domain. In embodiments, the signaling domain and the co-stimulatory signaling domain are located on different molecules in the same cell.

[0264] Embodiments relate to polynucleotides encoding CAR molecules described herein. Embodiments relate to modified cells comprising CAR molecules described herein. In embodiments, the modified cells described herein are T cells. Embodiments relate to methods for causing T cell responses and / or stimulating anti-tumor responses of immunotherapy in subjects, the method comprising: administering to a subject an effective amount of a composition comprising a modified cell population described herein, or contacting the modified cells described herein with cells comprising solid tumor antigens. Embodiments relate to a method for stimulating an immune response, the method comprising: contacting a target cell comprising GCC with a modified cell described herein, thereby allowing an immune response comprising the release of cytokines by the modified cells.

[0265] Embodiments relate to the use of a composition described herein in a method for treating a subject having gastrointestinal cancer (GI tract or digestive tract cancer), the method comprising: administering to the subject an effective amount of a composition comprising a first population of cells comprising a first CAR that binds to a first antigen, and a second population of cells comprising GUCY2C that binds to a second CAR, wherein the first antigen comprises a cell surface molecule of a white blood cell (WBC).

[0266] Embodiments relate to the use of a composition described herein in a method for enhancing the anti-tumor efficacy of immunotherapy in a subject with gastrointestinal cancer, the method comprising: administering to the subject an effective amount of a composition comprising a first population of cells comprising a first CAR that binds to a first antigen, and a second population of cells comprising GUCY2C that binds to a second CAR, wherein the first antigen comprises a cell surface molecule of a white blood cell (WBC), and the anti-tumor efficacy of the cell population is enhanced in the subject compared to a subject administered an effective amount of the composition without the first cell population.

[0267] In embodiments, a composition described herein comprises a pharmaceutical composition described herein.

[0268] In embodiments, gastrointestinal cancer includes cancers associated with esophagus, stomach, small intestine and large intestine (colorectal cancer), liver and / or pancreas. In embodiments, gastrointestinal cancer includes primary cancer and metastatic cancer. In embodiments, cells are T cells, NK cells or dendritic cells. In embodiments, WBC are granulocytes, monocytes or lymphocytes. In embodiments, WBC are B cells. In embodiments, the cell surface molecules of WBC are CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cell surface molecules of WBC are CD19, CD20, CD22 or BCMA. In embodiments, the cell surface molecules of WBC are CD19. In an embodiment, the first CAR comprises a scFv that binds to CD19, and an intracellular domain comprising 4-1BB or CD28 and a CD3 zeta domain, and the second CAR comprises a scFv that binds to GUCY2C or SEQ ID NO: 166, and comprises 4-1BB or CD28 and a CD3 zeta domain. In an embodiment, the antigen binding domain of the first CAR comprises an amino acid sequence of SEQID NO: 101, and the antigen binding domain of the second CAR comprises one or more amino acid sequences of SEQ ID NO: 105-130. In an embodiment, the second cell population comprises a lentiviral vector encoding a dominant negative form of a second CAR and PD-1.

[0269] In an embodiment, the first cell population comprises a lentiviral vector encoding a first CAR and a therapeutic agent. In an embodiment, the therapeutic agent comprises a cytokine. In an embodiment, the cytokine is IL-6 and / or INF-γ. In an embodiment, the cytokine is at least one of IL-6, IL-12, TNF-α or IFN-γ.

[0270] Embodiments relate to polynucleotides encoding the first CAR and the second CAR molecules as described herein. Embodiments relate to vectors comprising polynucleotides as described herein. Embodiments relate to cells comprising vectors as described herein. Embodiments relate to compositions comprising cell populations as described herein. Embodiments relate to the use or method of the compositions described herein in eliciting or causing a T cell response, the use or method comprising administering an effective amount of the composition.

[0271] Embodiments relate to antibodies that bind to GCC, wherein the antibody comprises an LVR comprising an amino acid sequence of SEQ ID NO: 109, 113 or 121 and an HVR comprising an amino acid sequence of SEQ ID NO: 105 or 117. Embodiments relate to CARs comprising an antigen binding domain comprising an antibody that binds to GCC or a fragment thereof. In embodiments, the antibody comprises an LVR comprising an amino acid sequence of SEQ ID NO: 109 or 113 and an HVR comprising an amino acid sequence of SEQ ID NO: 105. In embodiments, the LVR comprises an amino acid sequence of SEQ ID NO: 113 or 121, and the HVR comprises an amino acid sequence of SEQ ID NO: 117. In embodiments, the HVR is connected to a human IgG chain constant region, and the human IgG is IgG1 or IgG3. In embodiments, the antibody or antibody fragment is conjugated to a cytotoxic agent, and the cytotoxic agent is a radioactive isotope or a toxin. In embodiments, the antibody is a scFv, and the LVR is connected to the HVR via a linker.

[0272] Embodiments relate to a CAR comprising an antigen binding domain comprising at least one amino acid sequence of SEQ ID NOS: 125-130, wherein the CAR binds to GCC. Embodiments relate to a CAR comprising an antigen binding domain comprising one of the amino acid sequences SEQ ID NO: 125, 126 or 127, wherein the CAR binds to GCC. Embodiments relate to polynucleotides encoding antibodies or antibody fragments or CARs that bind to GCC. Embodiments relate to modified cells comprising the polynucleotides described herein.

[0273] Embodiments relate to the use of polynucleotides or nucleic acids, vectors, CARs, modified cells, modified cell populations, compositions, pharmaceutical compositions, kits or methods described herein in treating a subject with cell therapy. Embodiments also include treating a subject that is a mammal, and optionally wherein the mammal is a human. In embodiments, the subject suffers from or is diagnosed with cancer. In embodiments, the use comprises inducing and / or enhancing a T cell response in a subject, and optionally wherein the subject is a mammal, and optionally wherein the mammal is a human. In embodiments, the subject suffers from or is diagnosed with cancer

[0274] Embodiments relate to isolated nucleic acids or polynucleotides encoding humanized antibodies or antigen-binding fragments thereof, wherein the humanized antibodies or antigen-binding fragments thereof comprise heavy chain variable domain (HVR) sequences having an amino acid sequence of SEQ ID NO: 105, 117, 146, 148, 150, 156, 158, 160 or a combination thereof and light chain variable domain (LVR) sequences having an amino acid sequence of SEQ ID NO: 109, 121, 152, 154, 162, 164, or a combination thereof. Embodiments relate to expression vectors comprising isolated nucleic acids or polynucleotides as described herein, the isolated nucleic acids or polynucleotides being operably linked to control sequences recognized by host cells transfected with the expression vectors. Embodiments relate to host cells comprising expression vectors. Embodiments relate to CARs comprising scFvs, the scFvs comprising HCV sequences and LCV sequences.

[0275] In an embodiment, the humanized antibody or antigen-binding fragment thereof comprises an HCV sequence having the amino acid sequence of SEQ ID NO: 105 or 117, or an LCV sequence having the amino acid sequence of SEQ ID NO: 109 or 121, or a combination thereof.

[0276] In an embodiment, the humanized antibody or antigen-binding fragment thereof comprises an HVR sequence having the amino acid sequence of SEQ ID NOs: 146, 148, 150, 156, 158 and 160, or an LVR sequence having the amino acid sequence of SEQ ID NOs: 148, 158 and 160: 152, 154, 162 and 164, or a combination thereof.

[0277] In an embodiment, the antigen-binding fragment described herein is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab)2 and diabody.

[0278] In an embodiment, the CAR comprises a scFv comprising an HCV sequence having the amino acids of SEQ ID NO: 105 or 117 and an LCV sequence having the amino acid sequence of SEQ ID NO: 109 or 121.

[0279] Embodiments relate to methods for treating subjects with various forms of cancer, as described herein. The method includes administering an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising a modified cell population, the modified cell population comprising a polynucleotide encoding a humanized anti-MSLN CAR and a polynucleotide encoding a secretable form of scFv blocking ADAM17 connected by a P2A sequence. On the one hand, inhibiting ADAM17 can enhance the anti-tumor activity of tumor immunotherapy. On the other hand, inhibiting ADAM117 can prevent the membrane surface of MSLN from being abnormally spliced ​​by ADAM17, so that MSLN on the surface of tumor cells can be recognized by MSLNCART cells. The method can enhance the efficacy of 5-FU drug therapy (for colorectal cancer). EGFR ligands are synthesized as transmembrane precursor proteins, most of which can be cut into free forms by ADAM17 from the membrane surface. The EGFR ligands that come out can bind to the EGFR on the tumor surface to form positive feedback, thereby promoting tumor growth, differentiation and metastasis. At the same time, ADAM17 can also cut CD62L, leading to terminal differentiation of T, B and NK cells. The method is no longer limited to anti-MSLN immunotherapy. Nevertheless, it can also be used for other targeted immunotherapies, and this approach can be used for any immune cell, such as CAR T, TIL, CAR-NK, etc. When the chemotherapy drug 5-FU (5-Fluorouracil) is used to treat colorectal cancer, the drug may activate ADAM17, causing more growth factor ligands to fall off the membrane surface, thereby enhancing the activation of growth factor receptors and ultimately promoting the growth of colorectal cancer cells. This therapy is severely resistant to chemotherapy. Therefore, the combined use of 5-FU chemotherapy and ADAM17 inhibition can enhance the efficacy of 5-FU. This combination approach can also be extended to other similar chemotherapy drugs.

[0280] Embodiments relate to modified cells comprising a polynucleotide or nucleic acid encoding an antibody that binds ADAM metallopeptidase domain 17 (ADAM17), wherein the antibody is secretable or attached to the membrane of the modified cell.

[0281] In an embodiment, the polynucleotide comprises a polynucleotide encoding at least one nucleic acid sequence of SEQ ID NOS: 167-170. In an embodiment, the antibody is a secretable scFv. In an embodiment, the polynucleotide comprises a polynucleotide encoding SEQ ID NO: 169 or 170. In an embodiment, the antigen binding molecule is a CAR or TCR.

[0282] The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present disclosure should not be interpreted in any way as being limited to the following exemplary embodiments and examples, but should be interpreted as covering any and all variations that become apparent due to the teachings provided herein.

[0283] example

[0284] Example 4: GCC CAR

[0285] The sequence encoding the extracellular region of GCC was cloned into the recombinant protein expression vector PTSE-His system. The recombinant plasmid was transfected into HEK293 cells to express the fusion protein (GCC-His). The expressed fusion protein was purified using a GE Histrap FF affinity chromatography column. SDS-PAGE analysis showed that GCC-His (antigen) was expressed, and due to the heterogeneity of glycosylation, the molecular weight was approximately 60-80KDa. GCC-His was analyzed by ELISA using the positive control antibody 5F9. The positive control antibody binds to GCC-His. The extracellular region of the recombinant GCC was retained.

[0286] Based on Baxter's λ recombinase system (λ-Int), a fully human heavy chain antibody library (library capacity 2x108) and a fully human light chain antibody library (library capacity 1.5x10 7 ) were recombined in bacteria to obtain a recombinant human antibody library (Fab) with a capacity of more than 1.2x10 10 . Using GCC-His as antigen, the above large-capacity recombinant human Fab antibody library was displayed and screened following the solid phase screening strategy of the classical phage antibody library. After several rounds of screening, hundreds of clones were identified (phage-ELISA) and analyzed. Several positive monoclonal antibodies binding to GCC with different sequences were obtained and are provided below.

[0287] A lentiviral vector encoding a single CAR molecule was generated and transfected with T cells, and the expression of CAR was confirmed by flow cytometry. The techniques related to the construction of cell culture and cytotoxic T lymphocyte assays can be found in "Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains," PNAS, March 3, 2009, vol. 106 No. 9, 3360-3365 and "Chimeric Receptors Containing CD137 Signal Transduction Domains Mediate Enhanced Survival of T Cells and Increased Antileukemic Efficacy In Vivo," Molecular Therapy, Aug. 2009, vol. 17 no. 8, 1453-1464, the entire contents of which are incorporated herein by reference.

[0288] Primary T cells were transduced with lentiviruses including various CARs to establish different CAR T cells (i.e., 6701, 6708, and 6709). These cells were obtained from healthy human donors. Slow viruses include nucleic acid sequences encoding CAR molecules and IRES-mCherry (green) constructs, which encode green fluorescence to confirm CAR expression. Technology related to cell culture, construction of lentiviral vectors, and flow cytometry can be found in "Treatment of Advanced Leukemia in Mice with mRNA-Engineered T Cells, HUMAN GENE THERAPY 22: 1575–1586 (December 2011)", which is incorporated herein by reference as a whole.

[0289] Each type of CAR T cell was co-cultured with the corresponding antigen-expressing substrate cells, and the response of the CAR T cells induced by the substrate cells was measured. CAR T cells were co-cultured with target tumor cells at a ratio of E:T (effector cells to target cells) 1:1, 3:1, 10:1 and 30:1 (ie, CAR T cells: target tumor cells) for 24 hours. The supernatant was collected and the release of IFN-γ was measured. In the tested CAR molecules and their corresponding substrate cells, IFN-γ release was observed in a variety of combinations of CAR molecules and antigen-expressing substrate cells. The sequences and corresponding antigens of these CARs are listed in Table 4. However, when CAR T cells expressing CAR molecules were co-cultured with wild-type substrate cells, IFN-γ release was not obvious. Techniques related to cell culture and construction of cytotoxic T lymphocyte assays can be found in “Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains,” PNAS March 3, 2009, Vol. 106, No. 9, which is incorporated herein by reference in its entirety.

[0290] Figure 3A and 3B Flow cytometry results showing CAR expression on T cells. The results in Figure 3 show that GCC antigen can induce rapid expansion of three CAR T cells (6701 (CAR PCT Publication No.: SEQ ID NO: 40 of WO2018064921), 6708 (CAR SEQ ID NO: 132), 6709 (CAR SEQ ID NO: 133)) that specifically recognize GCC. Figure 4A and 4B The flow cytometry results of CAR T cell activation after co-culture with the corresponding substrate using CD8 and CD137 as markers are shown. The results in Figure 4 show that under the stimulation of GCC antigen, CD8+CAR T cells that specifically recognize GCC antigen can be effectively activated.

[0291] Figure 5A and 5B The flow cytometry results of activation of CAR T cells marked by CD4 and CD40L after co-culture with corresponding substrate cells are shown. The results in Figure 5 show that under the stimulation of GCC antigen, CD4+CAR T cells that specifically recognize GCC antigen can be effectively activated.

[0292] Figure 6Shown are the cytokines released by CAR T cells after co-culture with the corresponding substrate cells. Figure 6 The results showed that under the stimulation of GCC antigen, CAR T cells that specifically recognize GCC antigen can release a large amount of cytokines.

[0293] Table 4 scFv and CAR sequences of three CAR T cells

[0294]

[0295] Fig. 9 The anti-tumor activity of 6708 and 6709CAR T cells is shown. The results show that after 6708 and 6709CAR T cells were co-cultured with ordinary 293T cells and tumor cells T84 expressing GCC antigen, it can be found that 6708 and 6709CAR T cells showed strong anti-tumor effects after contact with tumor cells T84, but did not kill 293T cells that did not express GCC antigen.

[0296] Humanized heavy and light chain regions were designed and tested, and several heavy and light chains were identified based on their binding abilities. In addition, 12 humanized heavy and light chain combinations (Tables 5 and 6) of two sequences derived from mouse antibodies (i.e., 4A8F7 scFv (SEQ ID NO: 125) and 1D2C5 scFv (SEQ ID NO: 127)) were subjected to preliminary analysis of antibody activity. Their remaining binding abilities (Tables 8 and 9) are shown, and the corresponding sequences are SEQ ID NOs: 146-165. These antibody chains are provided in Tables 5-6. Technology related to humanized mouse antibodies can be found in PCT Patent Publication No.: WO2018126369, which is incorporated herein by reference in its entirety.

[0297] Table 5: 4A8F7 humanized antibody combination

[0298]

[0299] Table 6: 1D2C5 humanized antibody combination

[0300]

[0301] Tables 7 and 8 show the degree of humanization and affinity analysis of the above antibody combinations.

[0302] Table 7 1D2C5VH, 1D2C5VH-1(VH-h1), 1D2C5VH-2(VH-h2), 1D2C5VH-3(VH-h3), 1D2C5VK, 1D2C5VK-1

[0303] (VK-h1), 1D2C5VK-2 (VK-h2) degree of humanization

[0304]

[0305] Results: Through humanization, 1D2C5 obtained 3 humanized VH chains and 2 humanized VK chains.

[0306] Table 81 Affinity analysis of D2C5 and antibody combinations 1-6 in Table 6

[0307] Affinity Ka Kd KD GUCY2C-1D2C5 <![CDATA[6.702x10 4 ]]> <![CDATA[5.529x10 -4 ]]> <![CDATA[8.249x10 -9 ]]> 1D2C5VH-h1+1D2C5VK-h1-IgG1 <![CDATA[7.123x10 4 ]]> <![CDATA[4.119x10 -4 ]]> <![CDATA[5.283x10 -9 <!-- 29 -->]]> 1D2C5VH-h1+1D2C5VK-h2-IgG1 <![CDATA[4.38x10 4 ]]> <![CDATA[7.175x10 -4 ]]> <![CDATA[1.638x10 -8 ]]> 1D2C5VH-h2+1D2C5VK-h1-IgG1 <![CDATA[7.077x10 4 ]]> <![CDATA[9.344x10 -4 ]]> <![CDATA[1.32x10 -8 ]]> 1D2C5VH-h2+1D2C5VK-h2-IgG1 <![CDATA[5.231x10 4 ]]> <![CDATA[1.992x10 -3 ]]> <![CDATA[3.808x10 -8 ]]> 1D2C5VH-h3+1D2C5VK-h1-IgG1 <![CDATA[4.595x10 4 ]]> <![CDATA[1.333x10 -3 ]]> <![CDATA[2.901x10 -8 ]]> 1D2C5VH-h3+1D2C5VK-h2-IgG1 <![CDATA[3.371x10 4 ]]> <![CDATA[3.402x10 -3 ]]> <![CDATA[1.009x10 -7 ]]>

[0308] The results showed that the three humanized 1D2C5-VH chains and the two humanized 1D2C5-VK chains were combined into six antibody combinations, and the affinity of each antibody combination was measured. It was found that VH-h1 / VK-h1 had the highest affinity and the highest degree of humanization.

[0309] Table 9 Affinity analysis of 4A8F7 and antibody combinations 2, 3, 5, and 6 in Table 5

[0310] Affinity Ka Kd KD GUCY2C-4A8F7 <![CDATA[6.921x10 4 ]]> <![CDATA[3.568x10 -4 ]]> <![CDATA[5.156x10 -9 ]]> VH-h2+VK-h1 <![CDATA[5.689x10 4 ]]> <![CDATA[1.914x10 -3 ]]> <![CDATA[3.383x10 -8 ]]> VH-h2+VK-h2 <![CDATA[6.41x10 4 ]]> <![CDATA[1.864x10 -3 ]]> <![CDATA[2.908x10 -8 ]]> VH-h3+VK-h1 <![CDATA[6.061x10 4 ]]> <![CDATA[5.049x10 -3 ]]> <![CDATA[8.33x10 -8 ]]> VH-h3+VK-h2 <![CDATA[6.644x10 4 ]]> <![CDATA[5.764x10 -3 ]]> <![CDATA[8.674x10 -8 ]]>

[0311] The results showed that 3 humanized 4A8F7-VH chains and 2 humanized 4A8F7-VK chains were combined into 6 antibody combinations, and the affinity of each antibody combination was measured. It was found that only 4 combinations had an affinity of 10 -8 , the affinity is weaker than that of 4A8F7.

[0312] Table 10: Epitope analysis of 4A8F7vK and 1D2C5 antibodies

[0313]

[0314] The results showed that 5F9 could partially block the 4A8F7 / 1D2C5 binding signal (30% to 50% blocking effect), and there was no clear binding blocking effect between other antibodies, indicating that the epitopes of 5F9 and 4A8F7 partially overlapped; the epitopes of 5F9 and 1D2C5 partially overlapped; and the epitopes of 4A8F7 and 1D2C5 were completely different.

[0315] Table 11 below provides exemplary sequences. In addition, related sequences, compositions and methods for treating cancer are described in the following publications: WO2016138846, WO2018126369, WO2017167217, WO2019140100, WO2020146743, WO2021216731, WO2020106843, WO2020047306, WO2022150831, US20210060069 and US20210100841, which are incorporated by reference in their entirety.

[0316] Table 11 Sequence and description

[0317]

[0318]

[0319]

[0320] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety, just as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing has been described in terms of various embodiments, those skilled in the art will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.

Claims

1. A chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain, and wherein: The extracellular domain binds guanylate cyclase C (GCC) and comprises the amino acid sequence of SEQ ID NO:127 or 125.

2. The CAR of claim 1, wherein the extracellular domain binds to GCC and the CAR comprises the amino acid sequence of SEQ ID NO: 132 or 133.

3. The CAR according to claim 1 or 2, wherein the intracellular domain comprises a CD3zeta signaling domain and one or more co-stimulatory signaling regions, and the co-stimulatory signaling region comprises an intracellular domain of a co-stimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

4. An isolated nucleic acid encoding the CAR of any one of claims 1-3. A vector comprising the isolated nucleic acid of claim 4 .

6. A modified cell comprising the isolated nucleic acid of claim 4.

7. The modified cell of claim 6, wherein the modified cell further comprises one or more of the following: Nucleic acids encoding dominant negative forms of immune checkpoint molecules or their receptors with inhibitory effects, and the immune checkpoint molecules include programmed death receptor 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T lymphocyte attenuator (BTLA), T cell immunoglobulin mucin 3 (TIM-3), lymphocyte activation protein 3 (LAG-3), T cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4) or CD160; Endogenous TRAC gene expression is reduced; A nucleic acid encoding Htert or a nucleic acid encoding SV40LT, or a combination thereof; a nucleic acid encoding a suicide gene; Nucleic acids encoding IL-6, IFNγ, IL-12 and / or IL-2.

8. A composition comprising a cell population containing the isolated nucleic acid of claim 4 or the vector of claim 5, or a composition comprising the modified cell population of claim 6 or 7.

9. The composition of claim 8, wherein the cell population comprises lymphocytes comprising T cells, NK cells, macrophages or dendritic cells.

10. The composition of claim 9, wherein the lymphocytes are T cells and at least a portion of the T cells comprise a CAR that binds to a cell surface molecule of a white blood cell (WBC).

11. The composition according to claim 10, wherein: WBCs are B cells; The cell surface molecule of WBC is CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13; or The cell surface molecules of WBC are CD19, CD20, CD22 or BCMA.

12. The composition of any one of claims 8-11, wherein the composition comprises a pharmaceutical composition.

13. A multispecific binding molecule (PBM), wherein the PBM comprises at least one first binding domain that binds to a T cell and at least one second binding domain, wherein the second binding domain comprises the amino acid sequence of SEQ ID NO: 127 or 125.

14. The PBM of claim 13, wherein the first binding domain comprises a scFv that binds CD3.

15. The PBM according to claim 13 or 14, wherein the PBM is a bispecific antibody.

Images