Methods and compositions for expressing chimeric intracellular signaling molecules

Abstract

The name of the invention is methods and compositions of cells expressing chimeric intracellular signaling molecules. The invention relates to compositions and methods for enhancing T cell metabolism and activity for more efficient adoptive T cell therapy. By expressing intracellular signaling molecules in T cells, the T cells are metabolically enhanced, which have improved cytotoxicity and resistance to immunosuppression imposed by the tumor microenvironment. One aspect includes modified T cells and pharmaceutical compositions comprising the modified cells for use in adoptive cell therapy and treatment of diseases or conditions associated with enhanced immunity.

Description

[0001] This application is a divisional application. The original application was filed on August 25, 2016, with application number 2016800621428, and the invention title was "Method and Composition for Expressing Chimeric Intracellular Signal Transduction Molecules in Cells".

[0002] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 211,295, filed August 28, 2015, under 35 USC § 119(e), which is incorporated herein by reference in its entirety.

[0003] Statement regarding federally funded research or development This invention was completed with government support under CA120409-07 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention. Background Technology

[0004] During tumor growth, rapid growth can lead to insufficient blood supply, resulting in areas within the tumor with significantly lower oxygen concentrations than healthy tissue. The hypoxic microenvironment in solid tumors is a consequence of rapidly proliferating tumor cells consuming available oxygen, thus limiting the amount of oxygen available for further diffusion into the tumor tissue. To support continued growth and proliferation in challenging hypoxic environments, cancer cells have developed mechanisms that alter their metabolism. Consequently, hypoxia is associated with increased tumor resistance to chemotherapy and radiation therapy.

[0005] Many conventional reactive chemotherapy therapies fail to penetrate hypoxic regions far from vascular locations. Furthermore, chronic hypoxia restricts cancer cell proliferation, making dormant tumor cells in hypoxic regions of the tumor less susceptible to conventional antiproliferative agents—which typically actively target dividing cells closely adjacent to blood vessels. Under hypoxic conditions, genomic instability can lead to tumor cell variants that can survive in hypoxic environments through clonal selection and expansion. This clonal expansion can result in clinically detrimental tumor progression, metastasis, acquired resistance to chemotherapy, and treatment failure.

[0006] Given the role of hypoxia in tumor progression, metastasis, and resistance to therapy, as well as eventual treatment failure, there is a need for more effective methods to combat therapy-resistant cancer cells. This invention addresses this need. Summary of the Invention

[0007] As described herein, the present invention relates to compositions, methods, and uses of metabolically enhanced T cells. One aspect of the invention includes metabolically enhanced T cells comprising chimeric intracellular signaling molecules. The chimeric intracellular signaling molecules include intracellular domains of co-stimulatory molecules and are substantially lacking extracellular ligand-binding domains. Metabolically enhanced T cells express the chimeric intracellular signaling molecules.

[0008] In another aspect, the present invention includes metabolically enhanced T cells comprising chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules include an intracellular domain of a co-stimulatory molecule and a non-antigen-binding extracellular domain, the non-antigen-binding extracellular domain including the Fc of IgD or IgA. Metabolically enhanced T cells express the chimeric intracellular signaling molecules.

[0009] In another aspect, the present invention includes a method for generating metabolically enhanced T cells. The method includes introducing a nucleic acid sequence encoding a chimeric intracellular signaling molecule into a T cell. The nucleic acid sequence comprises a nucleic acid sequence of an intracellular domain of a co-stimulatory molecule and substantially lacks an extracellular ligand-binding domain. At least one co-stimulatory molecule on the T cell is stimulated. The stimulation activates the chimeric intracellular signaling molecule, thereby metabolically enhancing the T cell.

[0010] Another aspect of the invention includes a metabolically enhanced population of T cells comprising chimeric intracellular signaling molecules. These chimeric intracellular signaling molecules include intracellular domains of co-stimulatory molecules and are substantially lacking extracellular ligand-binding domains.

[0011] In various embodiments of the invention described herein, or any other aspects thereof, the expression of chimeric intracellular signaling molecules metabolically enhances T cells. In another embodiment, the expression of chimeric intracellular signaling molecules improves cytotoxicity and resistance to immunosuppression in the tumor microenvironment.

[0012] In yet another embodiment, the metabolically enhanced T cells further comprise a bispecific antibody. In another embodiment, the bispecific antibody comprises a first antigen-binding domain binding to a first antigen and a second antigen-binding domain binding to a second antigen. In yet another aspect, the first antigen-binding domain of the bispecific antibody binds to a target cell and the second antigen-binding domain binds to an activated T cell. In yet another embodiment, the bispecific antibody comprises an antigen-binding domain including first and second single-chain variable fragment (scFv) molecules.

[0013] Another embodiment includes a pharmaceutical composition comprising the metabolically enhanced T cells of the present invention and a pharmaceutically acceptable carrier. Yet another embodiment includes the use of the metabolically enhanced T cells of the present invention in the manufacture of a medicament for treating an immune response in a subject requiring it. Still another embodiment includes a method of treating a disease or condition in a subject associated with enhanced immunity, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the metabolically enhanced T cells of the present invention. Another embodiment includes a method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the metabolically enhanced T cells of the present invention. Yet another embodiment includes a method for stimulating a T cell-mediated immune response against target cells or tissues in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the metabolically enhanced T cells of the present invention.

[0014] In some embodiments of the methods claimed herein, the nucleic acid sequence is selected from DNA and mRNA. In some embodiments, introducing the nucleic acid sequence includes electroporating mRNA into T cells. In other embodiments, the nucleic acid sequence is a vector selected from plasmid vectors, viral vectors, retrotransposons, site-directed insertion vectors, and suicide expression vectors.

[0015] Some embodiments further include equipping T cells with bispecific antibodies, wherein the bispecific antibodies are displayed on the surface of the T cells. In some embodiments, equipping T cells includes contacting the T cells with the bispecific antibodies. In other embodiments, the bispecific antibodies specifically bind to the T cells. In still other embodiments, equipping T cells includes equipping T cells with two or more bispecific antibodies and the T cells display two or more bispecific antibodies. In yet other embodiments, two or more bispecific antibodies specifically bind to the T cells. In some embodiments, the bispecific antibodies comprise a combination of antibodies selected from: anti-CD3, anti-IgD Fc, and anti-IgA Fc.

[0016] In other embodiments, the bispecific antibody is chemically heteroconjugated to a polyclonal antibody specific to a tumor-associated antigen (TAA), and the T cell specifically binds to the TAA polyclonal antibody. In another embodiment, the T cell is equipped with a nucleic acid sequence encoding the bispecific antibody via electroporation. In yet another embodiment, the bispecific antibody includes an antigen-binding domain comprising a first intact immunoglobulin molecule and a second intact IgG immunoglobulin molecule. In some embodiments, at least one of the first or second intact immunoglobulin molecules includes IgG, IgA, or IgD.

[0017] Another embodiment of the invention includes a composition comprising metabolically enhanced T cells generated according to the methods described herein. Attached Figure Description

[0018] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangement and tools of the embodiments shown in the drawings.

[0019] Figure 1 This is a set of figures showing how hypoxia differentially affects CAR T cell survival depending on the co-stimulatory intracellular endodomain.

[0020] Figure 2 This is a graph showing the sea horse assay used to measure the metabolic characteristics of modified T cells. OCR – mitochondrial respiration via oxidative phosphorylation; ECAR – glycolysis of ATP; Spare Respriatory Capacity is associated with enhanced survival and persistence under stress.

[0021] Figure 3 This is a diagram showing the metabolic reprogramming of modified T cells expressing CARs with CD28 or 4-1BB signaling domains.

[0022] Figure 4 This is an illustration of T cells equipped with bispecific antibodies (Fab CD3 with Fab anti-target A or B) – which have CARs introduced via lentiviral vectors. The data utilizes intact IgG molecules with permanent Fc-Fc covalent bonds.

[0023] Figure 5 This is an illustration of a T cell equipped with bispecific antibodies—which has a CAR introduced via mRNA electroporation.

[0024] Figure 6 This refers to the performance of unequipped or equipped CART19 cells and non-CART19 cells (ATC) against cell lines expressing HER2 and EGFR, in contrast to those equipped with anti-HER2 bispecific antibody (HER2Bi) and anti-EGFR bispecific antibody (EGFRBi). 51 A graph group depicting cytotoxicity in Cr release assays. Using equipped or unequipped CART19 cells at an effector / target ratio of 10:1, targeting HER2-expressing SK-BR-3, EGFR-expressing HCT-8, or CD19. + / CD20 + (Daudi) and CD19 - / CD20 - The (U266) cell line was tested in triplicate for 18 hours to measure cytotoxicity.

[0025] Figure 7 It is displayed 51 A panel of graphs showing the relative cytotoxicity of equipped and unequipped CART19 cells measured during repeated exposure to the SK-BR-3 tumor target (HER2 and EGFR-positive breast cancer cell lines) at a 10:1 effector / target ratio in a Cr release assay at days 1, 4, 10, 18, and 25. The right panel shows the fold change in CART19 cell proliferation during repeated killing assays. The mean fold change in proliferation of equipped cells compared to unequipped cells after exposure to the SK-BR-3 target at days 1, 4, 10, 18, and 25. Cell counts and viability were assessed using trypan blue at indicated days during the 4-week culture period. Repeated cytotoxicity occurred in the presence of an appropriate tumor-associated antigen (TAA) (left panel), and proliferation occurred when appropriate engagement (HER2Bi) was present on CART cells.

[0026] Figure 8A This is a graph showing the cytotoxicity ratio of unequipped or HER2Bi- and EGFRBi-equipped CART19 cells to HER2 and EGFR-expressing breast cancer cells. The triplicate mean represents the results of plates seeded with equipped or unequipped cells at a 10:1 effector / target ratio on HER2 and EGFR-expressing breast cancer cell lines 18 hours prior. 51 Cytotoxicity of Cr release assay.

[0027] Figure 8B This graph shows the cytotoxicity ratio of unequipped or HER2Bi- and EGFRBi-equipped CART19 cells to HER2 and EGFR-expressing lung cancer cells. Equipped or unequipped CART19 cells were seeded in triplicate plates at an effector / target ratio of 10:1 on HER2 and EGFR-expressing lung cell lines for 18 hours. 51 Cytotoxicity of Cr release assay.

[0028] Figure 8C This graph shows the cytotoxicity of unequipped or HER2Bi- and EGFRBi-equipped CART19 cells versus non-CART19 cells (ATC) against HER2 and EGFR-expressing prostate / ovarian cancer cells. Equipped or unequipped CART19 cells were seeded in triplicate plates at an effector / target ratio of 10:1 on HER2 and EGFR-expressing cell lines for 18 hours. 51 Cytotoxicity of Cr release assay.

[0029] Figure 8D This graph shows the cytotoxicity ratio of unequipped or HER2Bi- and EGFRBi-equipped CART19 cells to non-CART19 cells (ATC) against HER2 and EGFR-expressing pancreatic cancer cells. Equipped or unequipped CART19 cells were seeded in triplicate plates at an effector / target ratio of 10:1 on HER2 and EGFR-expressing cell lines for 18 hours. 51 Cytotoxicity of Cr release assay.

[0030] Figure 8E This graph shows the cytotoxicity of unequipped or HER2Bi- and EGFRBi-equipped CART19 cells and non-CART19 cells (ATC) against melanoma / osteosarcoma / glioma cancer cells expressing HER2 and EGFR. Equipped or unequipped CART19 cells were seeded in triplicate plates at an effector / target ratio of 10:1 on HER2 and EGFR-expressing cell lines for 18 hours. 51 Cytotoxicity of Cr release assay.

[0031] Figure 9 This is a set of figures showing the expression of activating and inhibitory co-receptors on CD4 and CD8 T cell populations under conditions of depleted (cytotoxic), short-term (24 h), or long-term (120 h) antigen exposure to unequipped or equipped CART19 cells.

[0032] Figure 10 This is a diagram illustrating the sequence and basic structure of a chimeric intracellular signaling molecule, such as 4-1BBz. An example of a chimeric intracellular signaling molecule includes a fusion protein with a detectable marker, a hinge / transmembrane domain, and an intracellular domain.

[0033] Figure 11 This is an illustration of chimeric intracellular signaling molecules exhibiting myristylation signals. Chimeric intracellular signaling molecules that are undetectable on the cell surface will be detected using alternative markers, such as fluorescent proteins or cell surface molecules associated with expression via T2A or IRES elements.

[0034] Figure 12 This is an illustration of chimeric intracellular signaling molecules with fusion markers for detection. Optionally, detectable alternative markers are co-expressed using the T2A or IRES system, both of which are detectable markers for chimeric intracellular signaling molecules.

[0035] Figure 13 This is an illustration of T cells equipped with bispecific antibodies that contain chimeric intracellular signaling molecules introduced via a lentiviral vector.

[0036] Figure 14 This is an illustration of T cells equipped with bispecific antibodies that contain chimeric intracellular signaling molecules introduced via mRNA electroporation.

[0037] Figure 15 This diagram illustrates the enhanced ability of T cells equipped with bispecific antibodies to kill target cells. The tumor microenvironment enhances this effect by releasing inhibitory regulatory T cells (T cells). regs The Th1 cytokines and chemokines of the myeloid-derived repressive cells (MDSCs) population are altered, and endogenous immune cells are recruited to become cytotoxic memory effector cells and memory B cells that produce antibodies targeting tumors.

[0038] Figure 16 This is a set of images showing that when T cells equipped with bispecific antibodies are bound to a tumor target, irradiating these T cells blocks the allogeneic response (left image) but does not inhibit cytotoxicity (right image). "r" represents effector cells, and "s" represents stimulator cells. Indicates the irradiated cells.

[0039] Figure 17 This demonstrates metabolically enhanced T cells, whether unequipped or equipped with anti-CD20 bispecific antibody (CD20Bi), anti-EGFR bispecific antibody (EGFRBi), anti-GD2 bispecific antibody (GD2Bi), and anti-HER2 bispecific antibody (HER2Bi), targeting breast cancer and pancreatic / colorectal cancer cell lines expressing HER2 and EGFR. 51 A graph group showing the cytotoxicity ratio in the Cr release assay. Cytotoxicity was measured using equipped or unequipped T cells in triplicate for 18 hours at an effector / target ratio of 10:1.

[0040] Figure 18 This is a set of graphs showing the cytokine profile in the culture supernatant. Unequipped or equipped T cells or non-metabolic enhanced ATCs were seeded overnight in the absence of target cells or in the presence of target cells at an E:T = 10:1 (SKBR3). Values ​​are reported as pg / ml. The presence of cytokines (top) and chemokines (bottom) in the culture supernatant was determined by a Bio-Plex assay.

[0041] Figure 19A This is an expanded set of images showing T cells expressing flag-tagged chimeric intracellular signaling molecules—either CD8 transmembrane domains with 4-1BB and CD3ζ intracellular domains or membrane-anchored myristylated 4-1BB and CD3ζ intracellular domains.

[0042] Figure 19B This is a group of images showing enhanced CD4 expression in T cells expressing the chimeric intracellular signaling molecule tagged with a specific label. Normal donor ND317 T cells were activated using anti-CD3 / 28 beads and transduced to express the chimeric intracellular signaling molecule. These cells showed significant enhancement in the CD4 population.

[0043] Figure 19C This is an expanded set of images showing the transduction of 160 µl or 400 µl of virus into T cells with a tagged chimeric intracellular signaling molecule—possessing a 4-1 BB CD8 transmembrane domain and a CD3ζ intracellular domain. Normal donor ND317 T cells were transduced using the tagged chimeric intracellular signaling molecule. The percentage of T cells expressing the tagged chimeric intracellular signaling molecule decreased from day 6 to day 8 in the total population. '160v' means 160 µl virus / 1e at the initial transduction site. 6 Each cell.

[0044] Figure 20 This is a group of figures depicting the characterization of normal donor ND444 T cells transduced with high (50%) and low (15%) viral loads to express different percentages of chimeric intracellular signaling molecules. The detection of time-stamped T cells was lost.

[0045] Figure 21A This is a set of figures showing the population doubling and expansion of cell size of normal donor ND422 T cells labeled with transduction-tagged chimeric intracellular signaling molecules (BBz) or CAR19BBz. T cells were activated, transduced, and their expansion was monitored by a coulter counter, and similar profiles were shown until the cells became resting.

[0046] Figure 21B This is a set of figures showing CD4 and CD8 expression characterized by normal donor ND422 T cells on days 5 and 8, targeting the chimeric intracellular signaling molecule (BBz) or CAR19BBz tagged with the transduction tag. The total T cell population showed a bias towards CD4 expansion, without any additional enhancement observed in CD4+ cells via the chimeric intracellular signaling molecule. All data shown were gated to live CD3+ cells.

[0047] Figure 21C This is a group of graphs showing that the percentage of tagged T cells in normal donor ND422 T cells transduced with chimeric intracellular signaling molecules (BBz) or CAR19BBz did not decrease from day 5 to day 8. Detailed Implementation

[0048] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice to test the invention, preferred materials and methods are described herein. The following terms will be used in the description and claims of this invention.

[0049] It is also understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting.

[0050] This article uses the articles “a” and “a kind” to refer to one or more (i.e., at least one) grammatical objects of that article. For example, “an element” means one element or more elements.

[0051] As used herein, “approximately” when referring to a measurable value such as a quantity, period of time, etc., means to cover variations from a specified value of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, provided that such variations are suitable for the disclosed method.

[0052] As used in this article, “activation” refers to the state of T cells that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term “activated T cells” refers to T cells undergoing cell division, etc.

[0053] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; 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).

[0054] The term "antibody fragment" refers to a portion of a complete antibody and specifically to the antigenic determination variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0055] As used in this article, “antibody heavy chain” refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation.

[0056] As used in this article, "antibody light chain" refers to the smaller chain of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. α and β light chains refer to the two main isotypes of antibody light chains.

[0057] As used herein, "bispecific antibody" refers to an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly generated using the co-expression of two immunoglobulin heavy / light chain pairs. See, for example, Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be prepared using chemical bonding. See, for example, Brennan et al. (1985) Science 229:81. Bispecific antibodies comprise bispecific antibody fragments. See, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368.

[0058] As used herein, the term "synthetic antibody" means an antibody generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be interpreted as meaning an antibody generated from the synthesis of a DNA molecule that encodes an antibody and expresses an antibody protein, or that specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well-known in the art.

[0059] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule—indeed, all proteins or peptides—can act as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA—which includes nucleotide sequences or portions of nucleotide sequences encoding proteins that elicit an immune response—and therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be uniquely encoded by the full-length nucleotide sequence of a gene. It will be readily apparent that the invention includes, but is not limited to, the use of portions of nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response. Moreover, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that antigens can be generated, synthesized, or can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0060] As used herein, the term "antitumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. The "antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of this invention to prevent tumor formation at the first site.

[0061] As used herein, the term "arming" refers to displaying bispecific antibodies on the surface of cells. In one implementation, the bispecific antibody specifically binds to an antigenic epitope on a cell to be armed with the bispecific antibody—such as a T cell—and binds to another antigenic epitope, such as an antigenic epitope on a target cell—such as another type of cell.

[0062] According to the present invention, the term "autoantigen" means any autoantigen recognized as a foreign substance by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0063] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, type I diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Graves-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, thyroiditis, vasculitis, leukoplakia, myxedema, pernicious anemia, ulcerative colitis, etc.

[0064] As used in this article, the term "self" means any substance that originates from the same individual and is subsequently reintroduced into that individual.

[0065] "Also-alien" refers to grafts that originate from different animals of the same species.

[0066] "Heterogeneous" refers to grafts originating from animals of different species.

[0067] As used herein, "bispecificity" refers to a molecule that has binding specificity to at least two different binding epitopes. In one embodiment, the epitopes originate from the same binding partner. In another embodiment, the epitopes originate from two different binding partners. Molecules having bispecificity to different epitopes may include bispecific antibodies.

[0068] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In some embodiments, the cancer is medullary thyroid carcinoma.

[0069] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T-cell receptor engineered to express and specifically bind antigens on immune effector cells. CARs can be used as a therapy employing adoptive cell transfer. T cells are removed from a patient and modified to express receptors specific to a particular form of antigen. For example, in some embodiments, CARs have been expressed specifically for tumor-associated antigens. CARs may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain, the extracellular domain including a tumor-associated antigen-binding region. In some aspects, CARs comprise a fusion single-chain variable fragment (scFv)-derived monoclonal antibody fused to both the CD3-ζ transmembrane and intracellular domains. The specificity of the CAR design may be derived from a receptor ligand (e.g., a peptide). In some embodiments, by redirecting the specificity of T cells expressing a CAR specific to a tumor-associated antigen, the CAR can target cancer.

[0070] The term "chimeric intracellular signaling molecule" refers to a recombinant receptor that includes one or more intracellular domains of one or more co-stimulatory molecules. Chimeric intracellular signaling molecules are substantially lacking extracellular ligand-binding domains, such as non-antigen-binding domains, or are substantially lacking extracellular domains. In some embodiments, chimeric intracellular signaling molecules include additional domains, such as transmembrane domains, detectable labeling domains, and spacer domains.

[0071] As used herein, the term "conserved sequence modification" is intended to refer to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CDR region of an antibody can be replaced with other amino acid residues from the same side chain family, and the modified antibody's ability to bind antigens can be tested using the functional assays described herein.

[0072] As used herein, the term "co-stimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to associated co-stimulatory molecules on T cells, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation, in addition to the primary signals provided, for example, by binding the TCR / CD3 complex to a peptide-loaded MHC molecule. Co-stimulatory ligands may include, but are not limited to, CD7, 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 β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, in particular, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 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.

[0073] "Costimulatory molecules" refer to associated binding partners on T cells that specifically bind to costimulatory ligands to mediate costimulatory responses on T cells—such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2R γ,IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same functional capacity, and any combination thereof.

[0074] As used in this article, “co-stimulatory signal” refers to a signal combined with a primary signal, such as the TCR / CD3 linker, which leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0075] The term "cytotoxic" or "cytotoxic" refers to the killing or damage of cells. In one implementation, the cytotoxicity of metabolically enhanced cells is increased, such as increased cytolytic activity of T cells.

[0076] "Disease" is a state of health in which the animal is unable to maintain homeostasis, and in which, if the disease is not treated, the animal's health continues to deteriorate. In contrast, "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than when it was not in the state of the disorder. Without treatment, the disorder does not necessarily lead to a further decline in the animal's health.

[0077] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition as described herein that is effective in achieving a specific biological outcome or providing a therapeutic or preventative benefit. Such an outcome may include, but is not limited to, antitumor activity, as determined by any means suitable in the art.

[0078] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules possess either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties derived from them. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which serves as a template for the transcription of the gene or cDNA, can both be referred to as the protein or other product encoding that gene or cDNA.

[0079] As used in this article, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0080] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0081] As used herein, the term "expansion" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the number of ex vivo expanded T cells increases relative to the number originally present in the culture. In another embodiment, the number of ex vivo expanded T cells increases relative to the number of other cell types in the culture. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and multiplied outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0082] As used in this article, “expression” is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0083] "Expression vector" refers to a vector comprising a recombinant polynucleotide including an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as clomids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide.

[0084] As used herein, "homologous" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules—for example, two DNA molecules or two RNA molecules—or two polypeptide molecules. Two molecules are homologous when the subunit positions in both are occupied by the same monomeric subunits; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., five positions in a polymer of ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous. As applied to nucleic acids or proteins, as used herein, "homologous" refers to sequences having approximately 50% sequence identity. More preferably, the homologous sequences have approximately 75% sequence identity, and even more preferably, at least approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0085] Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the receptor's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and potency. In some cases, Fv frame region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Additionally, humanized antibodies may include residues not found in the receptor antibody or in the input CDR or frame sequence. These modifications are made to further improve and optimize antibody performance. Generally, humanized antibodies will include at least one and usually two variable domains, wherein all or almost all CDR regions correspond to those of non-human immunoglobulins and all or almost all FR regions are those of human immunoglobulin sequences. Humanized antibodies will also preferably include at least a portion of the immunoglobulin constant region (Fc), which is usually of human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0086] "Fully human" refers to immunoglobulins, such as antibodies, in which the complete molecule has an amino acid sequence of human origin or is composed of antibodies of the same human form.

[0087] As used herein, “identity” refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, such as two polypeptide molecules. Two amino acid sequences are identical when they have the same residues at the same positions; for example, if each of two polypeptide molecules has a position occupied by arginine, then they are identical at that position. In alignments, the identity or degree of identical residues at the same positions of two amino acid sequences is often expressed as a percentage. Identity between two amino acid sequences is a direct function of matching or identical positions; for example, if half the positions in two sequences (e.g., five positions in a polymer ten amino acids long) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., nine out of ten) are matching or identical, then the two amino acid sequences are 90% identical.

[0088] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, at the amino acid level or at the nucleic acid level, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95%, or even 99% identical to the sequence used for comparison.

[0089] The guide nucleic acid sequence can be complementary to one strand (nucleotide sequence) of the double-stranded DNA target site. The percentage of complementarity between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The guide nucleic acid sequence can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleotides long. In some embodiments, the guide nucleic acid sequence comprises a continuous stretch of 10 to 40 nucleotides. The variable targeting domain can consist of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, for example, the modifications described herein), or any combination thereof.

[0090] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program, from the Genetics Computing Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conserved substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 and e-100 The probability scores between them indicate closely related sequences.

[0091] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defending against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate anaphylaxis after exposure to an allergen by inducing the release of mediators from mast cells and basophils.

[0092] As used in this article, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes recognize an antigen molecule as a foreign substance and induce the formation of antibodies and / or activation of lymphocytes to remove the antigen.

[0093] As used herein, “instructional material” includes publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. Instructional material for the kits of the present invention may, for example, be attached to or shipped together with containers containing nucleic acids, peptides, and / or compositions of the present invention. Alternatively, the instructional material may be shipped separately from the containers, with the intention that the instructional material and the compounds be used in conjunction by the recipient.

[0094] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living organisms are not "separated," but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting substance is "separated." Separated nucleic acids or proteins can exist in a substantially purified form or can exist in unnatural environments, such as, for example, host cells.

[0095] As used in this article, "lentivirus" refers to a genus within the family Retroviridae. Among retroviruses, lentiviruses are the only ones capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral vectors provide a means to achieve significant levels of in vivo gene transfer.

[0096] The term "ligand" refers to a molecule, compound, or other binding partner that has a binding site—which has binding specificity to a ligand-binding domain. Ligands can also be referred to as "antigens."

[0097] As used herein, the term "modified" means an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

[0098] As used herein, the term "modulation" means mediating a detectable increase or decrease in the response level in a subject compared to the response level in a subject where no treatment or compound is present, and / or compared to the response level in a otherwise identical but untreated subject. The term encompasses disrupting and / or influencing natural signals or responses, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0099] In the context of this invention, the following abbreviations for common 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.

[0100] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate translations of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA phrase may also include introns, to the extent that the nucleotide sequence encoding that protein may contain one or more introns in some translations.

[0101] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For instance, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to that coding sequence. Typically, the operably linked DNA sequences are contiguous and, when necessary, join two protein-coding regions within the same reading frame.

[0102] The term “overexpressed” tumor antigen or “overexpression” of a tumor antigen is intended to refer to an abnormal level of expression of a tumor antigen in cells of a solid tumor within a specific tissue or organ of a disease region, such as a patient, relative to the expression level of normal cells from a tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be identified by standard tests known in the art.

[0103] "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0104] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Additionally, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides, as used herein, are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides, as used herein, include, but are not limited to, all nucleic acid sequences obtained by any means available in the art—non-limitingly including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR™, and by synthetic means.

[0105] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a sequence of proteins or peptides. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers; and longer chains, which are commonly referred to in the art as proteins, which have many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0106] As used herein, the term "promoter" is defined as a DNA sequence that is required for the specific transcription of a starting polynucleotide sequence and is recognized by the cellular synthetic machinery or introduced synthetic machinery.

[0107] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence operatively linked to a promoter / regulatory sequence required for the expression of a gene product. In some cases, this sequence may be a core promoter sequence, and in others, it may include enhancer sequences and other regulatory elements required for the expression of the gene product. For example, a promoter / regulatory sequence may be a sequence that expresses a gene product in a tissue-specific manner.

[0108] A "constitutive" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or defining a gene product, causes the production of that gene product in the cell under most or all physiological conditions.

[0109] An "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or defining a gene product, causes the gene product to be produced in the cell, essentially only when the inducer corresponding to the promoter is present in the cell.

[0110] "Tissue-specific" promoters are nucleotide sequences that, when operatively linked to a gene encoding a gene or a polynucleotide specified by the gene, cause the gene product to be produced in the cell essentially only if the cell is a cell of the tissue type corresponding to the promoter.

[0111] The term "resistance to immunosuppression" refers to the suppression of the lack of or activation of the immune system or the inhibition of the reduction of the activity or activation of the immune system.

[0112] "Signal transduction pathways" refer to the biochemical relationships between various signal transduction molecules—which play a role in transmitting signals from one part of the cell to another. The phrase "cell surface receptors" includes molecules and complexes of molecules that are capable of receiving signals and transmitting signals across the cell membrane.

[0113] "Single-chain antibody" refers to an antibody formed through recombinant DNA technology, in which segments of the immunoglobulin heavy and light chains are linked to the Fv region via an engineered span of amino acids. Various methods for generating single-chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; and Skerra et al. (1988) Science 242:1038-1041.

[0114] As used herein, the term "specifically binding" for antibodies means an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species also binds to that antigen from one or more species. However, such cross-species reactivity does not itself change the antibody class to specific. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not itself change the antibody class to specific. In some cases, the term "specifically binding" or "specifically binding" can be used to describe the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than generally recognizing and binding to proteins. If an antibody is specific to epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and an antibody will reduce the amount of labeled A bound to the antibody.

[0115] The term "stimulus" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its associated ligand, thereby mediating a signal transduction event—such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulus can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeleton.

[0116] "Stimulating molecule," as used in this article, means a molecule on a T cell that specifically binds to an associated stimuli ligand present on an antigen-presenting cell.

[0117] As used herein, "stimulatory ligand" means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to an associated binding partner (referred to herein as a "stimulatory molecule") on T cells, thereby mediating the primary response of T cells, including but not limited to activation, initiation of the immune response, and proliferation. Stimulatory ligands are well known in the art and encompass, in particular, MHC class I molecules loaded with peptides, anti-CD3 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD2 antibodies.

[0118] The term "object" is intended to include any living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, "object" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the object is a human.

[0119] As used herein, the term "substantially lacking extracellular domains" refers to a molecule that substantially lacks extracellular extruded domains. In one embodiment, the chimeric intracellular signaling molecule lacks any function performed via extracellular domains, such as antigen binding. In another embodiment, the chimeric intracellular signaling molecule includes transmembrane domains but lacks functional extracellular domains.

[0120] As used herein, the term "substantially lacking an extracellular ligand-binding domain" refers to a molecule that substantially lacks a binding domain that specifically binds to a molecule. In one embodiment, the chimeric intracellular signaling molecule lacks any functional ligand-binding domain in its extracellular domain, such as an antigen-binding domain. In another embodiment, the chimeric intracellular signaling molecule includes an extracellular domain but lacks the ability to specifically bind to a ligand, such as an antigen.

[0121] As used herein, “substantially purified” cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types—those normally associated with them in their natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term simply refers to cells that have been separated from cells normally associated with them in their natural state. In some embodiments, cells are cultured in vitro. In other embodiments, cells are not cultured in vitro.

[0122] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines the portion of the nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0123] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in T cell activation in response to antigen presentation. The TCR is responsible for recognizing antigens that bind to the major histocompatibility complex molecule. The TCR consists of a heterodimer of alpha (α) and beta (β) chains, but in some cells, it is composed of gamma and delta (γ / δ) chains. The TCR can exist in both α / β and γ / δ forms, which are structurally similar but have distinct anatomical locations and functions. Each chain consists of two extracellular domains—a variable and a constant domain. In some embodiments, the TCR can be modified on any cell including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0124] As used in this article, the term "therapeutic" means treatment and / or prevention. Therapeutic effects are achieved through the suppression, relief, or eradication of a disease state.

[0125] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which exogenous nucleic acids are transferred or introduced into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary target cells and their progeny.

[0126] "Treating" a disease, as used in this article, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0127] As used in this article, the term "tumor" refers to abnormally growing tissue, which can be benign, precancerous, malignant, or metastatic.

[0128] As used in this article, the phrases “under transcriptional control” or “operably linked” mean that the promoter is in the correct position and orientation associated with the polynucleotide to control the initiation of transcription and the expression of the polynucleotide by RNA polymerase.

[0129] A "vector" is a composition of matter comprising isolated nucleic acids and which can be used to deliver the isolated nucleic acids into cells. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0130] Scope: Throughout this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is used merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered as specifically disclosing all possible sub-ranges and individual numerical values ​​within those ranges. For example, a description of a range of 1 to 6 should be considered as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0131] describe This invention includes methods and compositions for enhancing T cell metabolism and activity to provide T cells that are more effective for adoptive T cell therapy. According to the invention, T cells are metabolically enhanced and possess improved cytotoxicity and resistance to immunosuppression imposed by the tumor microenvironment when they express chimeric intracellular signaling molecules or chimeric antigen receptors.

[0132] This invention also includes a combination method of adoptive cell therapy using bispecific antibodies (BiAbs) to equip metabolically enhanced T cells. Regulatory T cells (CD4+ / CD25) hi / CD127 lo ), granulocytes (CD14) - / HLA-DR - / CD11b + / CD33 + ) and monocytes (CD14) + / HLA-DR - / CD11b + / CD33 + The presence of myeloid-derived repressive cells (MDSCs) alters the tumor microenvironment to impair incoming immune effector cells. In vitro models have shown that T cells equipped with bispecific antibodies inhibit MDSC differentiation and attenuate T regulatory and MDSC repressive activity (Thakur A, et al., J Transl Med., 11:35, 2013). Bispecific antibody-equipped T cells also induce the secretion of Th1 cytokines, kill target cells, and extend post-tumor adaptation to transform the tumor microenvironment into a Th1 environment (Grabert, RC, et al., Clin. Canc. Res., 12:569-576, 2006) and immunize patients with their own tumor antigens.

[0133] Based on observations of these modified T cells, new modalities of T cell therapy have been developed and are described herein, which use either metabolically enhanced T cells alone or metabolically enhanced T cells that also encode BiAb to improve therapeutic efficacy.

[0134] Chimeric intracellular signal transduction molecules The present invention includes a chimeric intracellular signaling molecule within T cells. In one aspect, the invention includes a modified T cell comprising an isolated nucleic acid sequence encoding a chimeric intracellular signaling molecule, wherein the isolated nucleic acid sequence comprises a nucleic acid sequence of an intracellular domain of a co-stimulatory molecule and substantially lacks an extracellular ligand-binding domain, wherein the T cell expresses the chimeric intracellular signaling molecule.

[0135] In another aspect, the present invention includes modified T cells comprising chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules include intracellular domains of co-stimulatory molecules and are substantially lacking extracellular ligand-binding domains.

[0136] In another aspect, the invention includes cell populations comprising nucleic acids encoding chimeric intracellular signaling molecules that include intracellular domains and are substantially lacking extracellular ligand-binding domains, wherein the cell populations express the chimeric intracellular signaling molecules.

[0137] In one embodiment, the chimeric intracellular signaling molecule lacks any functional ligand-binding domains in its extracellular domain, such as an antigen-binding domain. In another embodiment, the chimeric intracellular signaling molecule includes an extracellular domain but lacks the ability to specifically bind to ligands or molecules. In yet another embodiment, the chimeric intracellular signaling molecule substantially lacks an extracellular domain.

[0138] In another aspect, the present invention includes metabolically enhanced T cells comprising chimeric intracellular signaling molecules comprising an intracellular domain of a co-stimulatory molecule and an extracellular domain comprising an antibody-binding domain—such as a single-chain fragment that includes a non-antigen-binding portion and lacks an antibody variable region or Fc portion, i.e., an extracellular domain of IgD or IgA.

[0139] In another aspect, the present invention includes metabolically enhanced T cells comprising chimeric intracellular signaling molecules and substantially lacking extracellular ligand-binding domains, the chimeric intracellular signaling molecules comprising intracellular domains of co-stimulatory molecules. The metabolically enhanced T cells express the chimeric intracellular signaling molecules. In some embodiments, the expression of the chimeric intracellular signaling molecules metabolically enhances the T cells. In some embodiments, the expression of the chimeric intracellular signaling molecules improves cytotoxicity and resistance to immunosuppression in the tumor microenvironment.

[0140] Intracellular domains The intracellular domain or other cytoplasmic domain of the chimeric intracellular signaling molecule of the present invention is responsible for activating cells in which the chimeric intracellular signaling molecule is expressed. The term "intracellular domain" therefore means an intracellular domain comprising any truncated portion sufficient to transduce the activation signal. In one embodiment, the intracellular domain includes a domain responsible for effector function. The term "effector function" refers to the specialized function of the cell. The effector function of T cells may, for example, include cytolytic activity or helper activity involving the secretion of cytokines.

[0141] In one embodiment, the intracellular domain includes a domain responsible for signal activation and / or transduction. The intracellular domain can transmit signal activation via protein-protein interactions, biochemical changes, or other responses to alter cell metabolism, shape, gene expression, or other cellular responses to the activation of chimeric intracellular signaling molecules.

[0142] In one embodiment, cells incorporating chimeric intracellular signaling molecules are metabolically enhanced. In another embodiment, cells incorporating chimeric intracellular signaling molecules exhibit improved cytotoxicity and resistance to immunosuppression, such as when the cells are in a tumor microenvironment.

[0143] Examples of intracellular domains used in this invention include, but are not limited to, the cytoplasmic portion of the T-cell receptor (TCR) and any co-stimulatory molecules that act in concert to initiate signal transduction after antigen-receptor binding, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0144] Examples of intracellular domains include fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same functional capacity, and any combination thereof.

[0145] In one embodiment, the intracellular domain of the chimeric intracellular signaling molecule includes any portion of a co-stimulatory molecule, such as at least one signaling domain derived from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), co-stimulatory molecules, any derivatives or variants of these sequences, any synthetic sequence having the same functional capability, and any combination thereof.

[0146] Other domains of chimeric intracellular signal transduction molecules Chimeric intracellular signaling molecules can include detectable markers. As used herein, the terms "protein marker," "detectable marker," or "marker" generally mean any oligopeptide or polypeptide linked to a chimeric intracellular signaling molecule for its detection. Markers can be attached to intracellular domains, hinge domains, transmembrane domains, or other domains of the chimeric intracellular signaling molecule. Markers can include up to 100 amino acids, between 10 and 50 amino acids, or between 5 and 25 amino acids. Markers can be removed or cleaved by the chimeric intracellular signaling molecule using chemical agents or enzymes such as proteases, inteptide splicing agents, peptidases, etc. Markers can also be used for affinity purification of chimeric intracellular signaling molecules. Examples of labels include, but are not limited to, chitin-binding proteins, maltose-binding proteins, thioredoxin-labeled proteins, fluorescein-labeled antibodies, glutathione S-transferase (GST), poly(His) labels, V5-labeled proteins, Myc-labeled proteins, HA-labeled proteins, biotin or biotin-like molecules, streptavidin-binding molecules, tag labels, or other labels known in the art.

[0147] Chimeric intracellular signaling molecules may include spacer domains. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions as a linker between any of its domains, such as linking a transmembrane domain to either an extracellular or cytoplasmic domain within a peptide chain. Spacer domains may be located at one or both ends of the chimeric intracellular signaling molecule. Spacer domains may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0148] In some embodiments, the chimeric intracellular signaling molecule further includes hinge and / or transmembrane domains. In one embodiment, the chimeric intracellular signaling molecule further includes hinge and / or transmembrane domains, such as the CD28 transmembrane domain and the CD8-α hinge domain. Examples of hinges and / or transmembrane domains include, but are not limited to, the following: α, β, or ζ chains of T cell receptors; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIR, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL2Rβ; IL2Rγ; IL7R. α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL , CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C and any combination thereof.

[0149] Bispecific antibodies This invention also covers bispecific antibodies. In one embodiment, the metabolically enhanced cells of this invention further comprise a bispecific antibody. A bispecific antibody comprises two distinct binding specificities and thus binds to two different antigens. In such an embodiment, the bispecific antibody comprises a first antigen-binding domain binding to a first antigen and a second antigen-binding domain binding to a second antigen. A bispecific antibody may specifically bind to more than one epitope on the same target, such as a cell or receptor, or specifically bind to more than one epitope on different targets. In another embodiment, the bispecific antibody comprises a bispecific antigen-binding domain.

[0150] This invention should not be construed as limited to any specific bispecific antibody. Rather, any bispecific antibody is useful in this invention. Bispecific antibodies can be constructed from synthetic antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFvs), single-domain antibodies, their antigen-binding fragments, and any combination thereof. In one embodiment, a bispecific antibody is constructed by linking two different antibodies or portions thereof—for example, Fab, F(ab')2, Fab', scFv, and sdAb from two different antibodies. Techniques for manufacturing human and humanized antibodies and antibody fragments such as scFvs are also described elsewhere herein. In another embodiment, the bispecific antibody includes an antigen-binding domain comprising first and second single-chain variable fragment (scFv) molecules.

[0151] Techniques for engineering and expressing bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537(1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be manufactured by: engineering the electrostatic steering effect (WO 2009 / 089004 A1) for manufacturing antibody Fc-heterodimer molecules; crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using “biantibody” techniques for manufacturing bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994); and the preparation of trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147: 60 (1991). Engineered antibodies having three or more functional antigen-binding sites—including “octopus antibodies”—are also included herein (see, for example, US 2006 / 0025576A1).

[0152] This invention includes bispecific antibodies having an antigen-binding domain that binds to target cells. In one embodiment, the bispecific antibody has specificity for a target cell antigen. In another embodiment, the bispecific antibody comprises an antibody or a fragment thereof that specifically binds to a target cell antigen. The target cell antigen may include the same target cell antigen that binds to a T cell receptor or may include different target cell antigens. The target cell antigen may include any type of ligand found on the surface of the target cell, including ligands on T cells (e.g., CD3, CD2, or other antigens expressed on T cell blasts). For example, a target cell antigen may be selected because it recognizes a ligand that acts as a cellular marker on a target cell associated with a specific disease state. Thus, examples of cellular markers that may act as ligands for the antigen portion domain of a bispecific antibody include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In one embodiment, the target cell antigen includes any tumor-associated antigen (TAA), any viral antigen, or any fragment thereof.

[0153] In another embodiment, the bispecific antibody has specificity for at least one antigen on T cells—such as metabolically enhanced T cells as described elsewhere herein. T cell antigens include antigens found on the surface of T cells. T cell antigens may include co-stimulatory molecules as described elsewhere herein. In one embodiment, the T cell antigen is CD3, CD4, CD8, the T cell receptor (TCR), or any fragment thereof. In this embodiment, the bispecific antibody comprises an antibody that specifically binds to a T cell antigen. Examples of bispecific antibodies include anti-CD3, anti-CD4, anti-CD8, anti-TCR, anti-IgD Fc, anti-IgA Fc, any fragment thereof, and any combination thereof. Other target antigens of the bispecific antibody may also be T cell antigens such as CD3, CD4, CD8, TCR, or any fragment thereof (e.g., an anti-CD3 x anti-CD3 construct). In another embodiment, the bispecific antibody is chemically heteroconjugated to a polyclonal antibody specific to a tumor-associated antigen (TAA), and the T cell specifically binds to the TAA polyclonal antibody.

[0154] Another embodiment of the invention includes metabolically enhanced T cells as described herein, wherein a first antigen-binding domain binds to a target cell and a second antigen-binding domain binds to an activated T cell.

[0155] Metabolically enhanced T cells, as described elsewhere herein, can be equipped with bispecific antibodies. When cells are equipped with bispecific antibodies, the cells come into contact with the bispecific antibodies, and the bispecific antibodies specifically bind to antigens on the cell surface via one of the antigen-binding domains of the bispecific antibody. In another embodiment, T cells are equipped with two or more bispecific antibodies, and the T cells exhibit two or more bispecific antibodies. In such an embodiment, the T cells specifically bind to at least two of the bispecific antibodies.

[0156] Alternatively, the cells can express and secrete bispecific antibodies. When cells express bispecific antibodies, the nucleic acid sequence encoding the bispecific antibody can be introduced into the cells. The nucleic acid sequence can be introduced by any method described elsewhere herein or by other methods known in the art. In one embodiment, the cells are electroporated using the nucleic acid sequence encoding the bispecific antibody.

[0157] Chimeric antigen receptor (CAR) In another aspect of the invention, metabolically enhanced T cells are generated by expressing a CAR therein. Therefore, the invention encompasses CARs and nucleic acid constructs encoding CARs, wherein the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0158] One or more domains or fragments of domains of a CAR can be human. In one embodiment, the invention includes a fully human CAR. The nucleic acid sequence encoding the desired domain can be obtained using recombination methods known in the art, such as by screening a library from cells expressing the gene using standard techniques, by deriving the gene from a vector known to include it, or by directly isolating it from cells and tissues containing it. Alternatively, the gene of interest can be generated synthetically rather than as a cloned molecule.

[0159] In one aspect, the present invention includes metabolically enhanced tumor-specific T cells comprising a chimeric antigen receptor (CAR) and a bispecific antibody, wherein the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule, and the bispecific antibody binds to a target on tumor cells and the T cells, and wherein the T cells exhibit improved cytotoxicity and resistance to immunosuppression at the solid tumor site. In one embodiment, the T cells include a nucleic acid sequence encoding the CAR, and optionally, a nucleic acid sequence encoding the bispecific antibody.

[0160] Antigen-binding domain In one embodiment, the CAR of the present invention includes an antigen-binding domain that binds to an antigen on a target cell. Examples of cell surface markers that can serve as antigens binding to the antigen-binding domain of the CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0161] The choice of antigen-binding domain depends on the type and number of antigens present on the surface of target cells. For example, antigen-binding domains can be selected to recognize antigens that act as cell surface markers on target cells associated with a specific disease state.

[0162] In one embodiment, the antigen-binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In one embodiment, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes.

[0163] The antigen-binding domain may include any domain that binds to an antigen and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Thus, in one embodiment, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof.

[0164] In some cases, it is advantageous for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be advantageous for the antigen-binding domain of the CAR to include human antibodies, humanized antibodies as described elsewhere in this document, or fragments thereof.

[0165] Another advantage is that the antigen-binding domain is operatively linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain—both described elsewhere herein—for expression in cells. In one embodiment, the nucleic acid encoding the antigen-binding domain is operatively linked to both a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the intracellular domain.

[0166] Transmembrane domain Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain that links the antigen-binding domain of the CAR to an intracellular domain. In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0167] The transmembrane domain can be derived from natural or synthetic sources. When the source is natural, the domain can originate from any membrane-binding or transmembrane protein. Specifically, the transmembrane region used in this invention can be derived from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least one or more of the transmembrane regions mentioned above). In some cases, various human hinges, including human Ig (immunoglobulin) hinges, may also be used.

[0168] In one embodiment, the transmembrane domain may be synthetic, in which case it will include dominant hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0169] Intracellular domains The intracellular domains or other cytoplasmic domains of CAR include intracellular domains similar to or the same as those of chimeric intracellular signaling molecules described elsewhere in this document, and are responsible for activating cells in which CAR is expressed.

[0170] In one implementation, the intracellular domains of the CAR include domains responsible for signal activation and / or transduction.

[0171] Examples of intracellular domains used in this invention include, but are not limited to, the cytoplasmic portion of the T-cell receptor (TCR) and any co-stimulatory molecules that act in concert to initiate signal transduction after antigen-receptor binding, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0172] Examples of intracellular domains include fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequence of costimulatory molecules with the same functional capacity, and any combination thereof.

[0173] In one embodiment, the intracellular domain of the CAR includes any portion of a co-stimulatory molecule, such as at least one signal transduction domain derived from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0174] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link any domain, such as the transmembrane domain, to the antigen-binding domain or the intracellular domain in a polypeptide chain. The spacer domain may be located at one or both ends of the CAR. In one embodiment, the spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and more preferably 25 to 50 amino acids. In another embodiment, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the intracellular domain of the CAR. Examples of linkers include glycine-serine duplexes.

[0175] Human antibodies When using the antigen-binding domain of a bispecific antibody or CAR, a human antibody or fragment thereof may preferably be used. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be manufactured by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645, WO98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO91 / 10741; each of which is incorporated herein by reference in its entirety. Bispecific antibodies may also include antibodies in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources.

[0176] Human antibodies can also be generated using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be randomly or through homologous recombination introduced into mouse embryonic stem cells. Optionally, in addition to human heavy chain and light chain genes, human variable, constant, and polymorphic regions can also be introduced into mouse embryonic stem cells. Through homologous recombination, mouse heavy chain and light chain immunoglobulin genes can be defunctionalized, either alone or simultaneously with the introduction of human immunoglobulin gene loci. For example, homozygous deletion of the antibody heavy chain J region (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with a selected antigen—for example, all or part of the polypeptide of the present invention. Antibodies against the selected target can be obtained from the immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes from transgenic mouse harbors undergo rearrangement during B-cell differentiation and subsequently class switching and somatic mutations. Therefore, using such a technique, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3. For a review of this technique for generating human antibodies, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)). For a detailed discussion of this technology for generating human antibodies and human monoclonal antibodies, and the protocols for producing such antibodies, see, for example, PCT Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated herein by reference in its entirety. Furthermore, contracts may be entered into with companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) to provide human antibodies against selected antigens using technologies similar to those described above.For a specific discussion of how transferring human germline immunoglobulin gene arrays into germline mutant mice after antigen challenge will lead to the production of human antibodies, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0177] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from all components of immunoglobulin variable (V) domain genes from non-immunized donors. According to this technique, antibody V domain genes are cloned in frame-compliant fashion into the major or minor coat protein genes of filamentous phages such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of the phage genome, the selection based on the functional properties of antibodies also leads to the selection of genes encoding antibodies that display those properties. Thus, phages mimic some properties of B cells. Phage display can be performed in various forms; for a review, see, for example, Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene segments from several sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated antibodies from a small, randomly combined library of the V gene derived from the spleen of unimmunized mice. Diverse arrays of zosadone antibodies. Antibodies can be constructed from all components of the V gene from unimmunized human donors, and diverse arrays of antigens (including autoantigens) can be isolated generally following techniques described below: Marks et al., J. Mol. Biol., 222:581-597 (1991) or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

[0178] Human antibodies can also be generated in vitro from activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety). Human antibodies can also be generated in vitro using hybridoma techniques, such as, but not limited to, those described by Roder et al. (Methods Enzymol., 121:140-167(1986)).

[0179] Humanized antibodies Optionally, in some embodiments, the nonhuman antibody may be humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring antibodies in humans. For example, in this invention, the antibody or a fragment thereof may include a nonhuman mammalian scFv. In one embodiment, the antigen-binding domain portion is humanized.

[0180] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR-transplantation (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering, or resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al, 1994, Protein Engineering, 7(6):805-814; and Roguska et al, 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain truncation (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety) and techniques disclosed in, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., ProteinEng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Typically, the framework residues in the framework region are replaced with corresponding residues from the CDR donor antibody to alter, preferably, the antigen binding.These framework substitutions were identified using methods well known in the art, such as by modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and by identifying uncommon framework residues at specific locations through sequence comparison (see, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

[0181] Humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “input” residues and are typically derived from an “input” variable domain. Thus, humanized antibodies comprise one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Humanization of antibodies is well known in the art and can generally be carried out following the approach of Winter and collaborators (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), which involves replacing rodent CDRs or CDR sequences with the corresponding human antibody sequences, i.e., CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; and US Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized chimeric antibodies, the variable domain, which is substantially smaller than that of a full-fledged human, has been replaced by a corresponding sequence from a non-human species. In fact, humanized antibodies are typically human antibodies in which some of the CDR residues and possibly some of the framework (FR) residues have been replaced by residues from similar sites in rodent antibodies. Humanization of antibodies can also be achieved by cladding or surface reconstruction (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain truncation (US Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0182] The selection of human variable domains—both light and heavy—used to manufacture humanized antibodies is aimed at reducing antigenicity. Following a so-called “best-fit” approach, the sequence of the variable domain of a rodent antibody is screened against an entire library of known human variable domain sequences. The human sequence that best matches the rodent sequence is then accepted as the human frame (FR) for humanized antibodies (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another approach uses specific frames derived from common sequences of all human antibodies from specific light or heavy chain subgroups. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety).

[0183] Antibodies can be humanized while retaining high affinity for target antigens and other favorable biological properties. According to one aspect of the invention, humanized antibodies are prepared by analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available to diagram and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examining these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequences, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind to target antigens. In this way, FR residues can be selected and combined from the acceptor and input sequences to achieve desired antibody properties, such as increased affinity for target antigens. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0184] Humanized antibodies retain similar antigen specificity to the original antibodies. However, using certain humanization methods, “directed evolution” approaches can be used to increase the binding affinity and / or specificity of antibodies to target antigens, as described by Wu et al., J. Mol. Biol., 294:151 (1999), the contents of which are incorporated herein by reference in their entirety.

[0185] carrier Vectors can be used to introduce chimeric intracellular signaling molecules or CARs into T cells as described elsewhere herein. In one aspect, the invention includes a vector comprising a nucleic acid sequence encoding a chimeric intracellular signaling molecule and optionally a nucleic acid sequence encoding a bispecific antibody as described herein. In another aspect, the invention includes a vector comprising a nucleic acid sequence encoding a CAR and optionally a nucleic acid sequence encoding a bispecific antibody as described herein. In one embodiment, the vector includes a plasmid vector, a viral vector, a retrotransposon (e.g., piggyback, sleeping beauty), a site-directed insertion vector (e.g., CRISPR, zinc finger nuclease, TALEN), or a suicide expression vector, or other vectors known in the art.

[0186] All the constructs mentioned above can be used with third-generation lentiviral vector plasmids approved for human cells, other viral vectors, or RNA. In one embodiment, the vector is a viral vector, such as a lentiviral vector. In another embodiment, the vector is an RNA vector.

[0187] The generation of any molecule described herein can be verified by sequencing. The expression of full-length proteins can be verified using immunoblotting, immunohistochemistry, flow cytometry, or other techniques well-known and available in the art.

[0188] This invention also provides vectors in which the DNA of this invention is inserted. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of resulting in low immunogenicity in the objects to which they are introduced.

[0189] Expression of natural or synthetic nucleic acids is typically achieved by operatively linking a nucleic acid or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors are typically capable of replicating in mammalian cells and / or integrating into the mammalian cellular genome. A typical vector contains transcription and translation terminators, an initial sequence, and a promoter, all useful for regulating the expression of the desired nucleic acid sequence.

[0190] Nucleic acids can be cloned into any number and type of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0191] Expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0192] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in a region 30–110 bp upstream of the start site, but recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, allowing the promoter to maintain function when an element is reversed or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can function cooperatively or independently to initiate transcription.

[0193] An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, elongation factor-1α promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch capable of turning on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turning off such expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0194] To assess the expression of peptides or their components, expression vectors introduced into cells can also contain either or both of optional marker genes or reporter genes to facilitate the identification and selection of expressing cells from cell populations seeking transfection or infection via viral vectors. In other respects, optional markers can be carried on separate segments of DNA and used in co-transfection procedures. Both optional markers and reporter genes can be side-linked with appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes, such as neo.

[0195] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is indicated by readily detectable properties such as enzymatic activity. The expression of the reporter gene is assessed at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well-known and can be prepared or commercially available using known techniques. Generally, constructs exhibiting the highest levels of reporter gene expression with minimal 5' flanking regions are identified as promoters. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0196] Introduction of nucleic acids Methods for introducing and expressing genes—such as chimeric intracellular signaling molecules or CARs—into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0197] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY. Commercially available methods can be used to introduce nucleic acids into target cells, including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany). Nucleic acids can also be introduced into cells in the following ways: using cationic liposome-mediated transfection, using lipid transfection, using polymer encapsulation, using peptide-mediated transfection, or using biological projectile particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

[0198] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. RNA vectors include vectors having an RNA promoter and / or other relevant domains for producing RNA transcripts. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0199] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane capsule).

[0200] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Liposome formulations are intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can associate with lipids. Liposome-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linkers associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, containing or complexed with micelles, or otherwise associated with lipids. Combinations of lipids, lipid / DNA, or lipid / expression vectors are not limited to any specific structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a “collapsed” structure. They can also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets—which are naturally present in the cytoplasm—as well as a class of compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0201] Suitable lipids are available from commercial sources. For example, dimyristicophosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; dimyristicophosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). The lipid stock solution in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general term encompassing a variety of single and multilayer lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicle structures containing a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess of aqueous solution. Before forming a closed structure and embedding water and dissolved solutes between the lipid bilayers, lipid components undergo self-rearrangement (Ghosh et al., 1991 Glycobiology 5: 505-10). However, this also includes compositions that have different structures in solution compared to normal vesicle structures. For example, lipids can present as micelle structures or exist simply as heterogeneous aggregates of lipid molecules. Lipid-transfected amine-nucleic acid complexes are also considered.

[0202] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the molecules described herein, various tests can be performed to confirm the presence of the nucleic acid in the host cells. Such tests include, for example, "molecular biology" tests well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and "biochemical" tests, such as those using immunological means (ELISA and protein blotting) or tests described herein to identify reagents falling within the scope of this invention to detect the presence or absence of a specific peptide.

[0203] In one embodiment, one or more nucleic acid sequences described elsewhere herein are introduced by a method selected from transduced cell populations, transfected cell populations, and electroporated cell populations. In one embodiment, the cell population comprises one or more nucleic acid sequences described herein.

[0204] In one implementation, the nucleic acid introduced into the cell is RNA. In another implementation, the RNA is mRNA comprising in vitro transcribed RNA or synthetic RNA. RNA is produced by in vitro transcription using a template generated using polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. The desired in vitro transcription template is a chimeric intracellular signaling molecule and / or a bispecific antibody.

[0205] PCR can be used to generate templates for in vitro transcribed mRNA—which is subsequently introduced into cells. Methods for performing PCR are well known in the art. Primers used for PCR are designed to have regions substantially complementary to a region of the DNA to be used as a PCR template. As used herein, “substantially complementary” means a sequence in which most or all bases of the primer sequence are complementary, or one or more bases are non-complementary or mismatched nucleotides. Substantially complementary sequences are capable of annealing or hybridizing with a target DNA DNA under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a gene (open reading frame) that is normally transcribed in a cell, including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene encoding a specific domain of interest. In one embodiment, primers are designed to amplify a coding region of human cDNA that includes all or part of the 5' and 3' UTRs. Primers suitable for PCR are generated using synthetic methods well known in the art. A “forward primer” is a primer containing a nucleotide region substantially complementary to a nucleotide on the DNA template upstream of the DNA sequence to be amplified. In this article, "upstream" refers to the 5' position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer containing a nucleotide region substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. In this article, "downstream" refers to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0206] Chemical structures that promote RNA stability and / or translation efficiency can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the length of the 5' UTR is between zero and 3000 nucleotides. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be altered by various methods, including but not limited to primers designed for PCR that anneal to different regions of the UTR. Using this method, those skilled in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency after transfection and transcription of the RNA.

[0207] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, non-endogenous UTR sequences for the gene of interest can be added by incorporating the UTR sequence into the forward and reverse primers or through any other modification of the template. Using non-endogenous UTR sequences for the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of transcribed RNA based on the properties of UTRs well known in the art.

[0208] In one embodiment, the 5' UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when adding a non-endogenous 5' UTR to a gene of interest via PCR as described above, a common Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the translation efficiency of some RNA transcripts, but do not appear to be necessary for efficient translation of all RNAs. The requirement for Kozak sequences of many mRNAs is known in the art. In other embodiments, the 5' UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to inhibit exonuclease degradation of the mRNA.

[0209] To enable RNA synthesis from a DNA template without gene cloning, a transcription promoter should be appended to the DNA template upstream of the sequence to be transcribed. When the sequence acting as the promoter of RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The common nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0210] In one implementation, the mRNA has a 5' cap and a 3' polyadenylated tail, both of which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatameric product unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces normal-sized mRNA, which is ineffective in eukaryotic transfection, even if it is polyadenylated post-transcriptionally.

[0211] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0212] The conventional method for integrating poly-A / T regions into DNA templates is molecular cloning. However, poly-A / T sequences integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning process not only laborious and time-consuming but also frequently unreliable. Therefore, methods that allow the construction of DNA templates with poly-A / T 3' regions without cloning are highly desirable.

[0213] The poly-A / T segment of the transcribed DNA template can be generated during PCR using a reverse primer containing a poly-T tail—for example, a 100-T tail (the size can be 50-5000 Ts)—or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The polyadenylated tail also provides stability to the RNA and reduces its degradation. Typically, the length of the polyadenylated tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the polyadenylated tail is between 100 and 5000 adenosine.

[0214] Following in vitro transcription, the polyadenylated tail of RNA can be further extended using polyadenylated polymerases, such as E. coli polyadenylated polymerase (E-PAP). In one embodiment, increasing the length of the polyadenylated tail from 100 nucleotides to between 300 and 400 nucleotides results in an approximately two-fold increase in RNA translation efficiency. Additionally, attaching different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the polyadenylated tail using polyadenylated polymerase. ATP analogs can further increase RNA stability.

[0215] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trendsin Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0216] The RNA generated by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that enables initiation of translation via a start cap-independent ribosome. It may include any solute suitable for cell electroporation, and may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0217] Several in vitro transcribed RNA (IVT-RNA) vectors are known in the literature, which are used as templates for in vitro transcription in a standardized manner and have been genetically modified in such a way that stable RNA transcripts are produced. Currently, the protocol used in this field is based on a plasmid vector having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by the gene of interest flanked by a non-translated region (UTR) at 3' and / or 5', and a 3' polyadenylated cassette containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylated cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylated cassette thus corresponds to the subsequent polyadenylated sequence in the transcript. As a result of this procedure, some nucleotides remain as partial enzyme cleavage sites after linearization and extend or mask the polyadenylated sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced from such constructs within cells.

[0218] In one aspect, RNA constructs are delivered into cells via electroporation. See, for example, formulations and methods for electroporating nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, and US 2004 / 0092907A1. Several parameters, including the electric field strength required for electroporating any known cell type, are generally known in the relevant research literature and in numerous patents and applications in this field. See, for example, US Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as the MedPulser™ DNA Electroporation Therapeutic System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Patent Nos. 6,567,694, 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482. Electroporation can also be used for in vitro cell transfection, for example, as described in US20070128708A1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Therefore, the electroporation-mediated administration of expression constructs including nucleic acids into cells using any of the many available devices and electroporation systems known to those skilled in the art provides exciting new means of delivering RNA of interest to target cells.

[0219] Generate metabolically enhanced T cells In one aspect, the present invention includes a method for generating metabolically enhanced T cells. The method includes introducing a nucleic acid sequence encoding a chimeric intracellular signaling molecule into a T cell, wherein the nucleic acid sequence includes a nucleic acid sequence of an intracellular domain of a co-stimulatory molecule and substantially lacks an extracellular ligand-binding domain. At least one co-stimulatory molecule on the T cell is stimulated, which activates the chimeric intracellular signaling molecule, thereby metabolically enhancing the T cell.

[0220] In one embodiment, the nucleic acid sequence is selected from DNA and mRNA. In another embodiment, the nucleic acid sequence is electroporated into T cells. The nucleic acid sequence can be a vector. Examples of vectors include, but are not limited to, plasmid vectors, viral vectors, retrotransposons, site-directed insertion vectors, and suicide expression vectors.

[0221] In some embodiments, the method for generating metabolically enhanced T cells further includes equipping T cells with bispecific antibodies, wherein the bispecific antibodies are displayed on the surface of the T cells. In one embodiment, equipping T cells includes contacting the T cells with the bispecific antibodies. In another embodiment, the bispecific antibodies specifically bind to the T cells. In yet another embodiment, T cells are equipped with two or more bispecific antibodies, and the T cells display two or more bispecific antibodies. In yet another embodiment, two or more bispecific antibodies specifically bind to the T cells. The bispecific antibodies may include a combination of antibodies selected from anti-CD3, anti-IgD Fc, and anti-IgA Fc. In some embodiments, the bispecific antibodies are chemically heteroconjugated to a polyclonal antibody specific to a tumor-associated antigen (TAA), and the T cells specifically bind to the TAA polyclonal antibody.

[0222] In some embodiments, the equipped cells include a nucleic acid sequence encoding a bispecific antibody via electroporation. The bispecific antibody includes a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen. In one embodiment, the bispecific antibody includes a first antigen-binding domain that binds to a target cell and a second antigen-binding domain that binds to an activated T cell. In another embodiment, the bispecific antibody includes an antigen-binding domain comprising first and second single-chain variable fragment (scFv) molecules. In yet another embodiment, the bispecific antibody includes an antigen-binding domain comprising a first intact immunoglobulin molecule and a second intact IgG immunoglobulin molecule. At least one of the first or second intact immunoglobulin molecules may be IgG, IgA, or IgD.

[0223] Optionally, in another aspect, the present invention includes a method for generating modified T cells, comprising electroporating a population of T cells using a nucleic acid sequence encoding a chimeric intracellular signaling molecule, wherein the nucleic acid sequence comprises a nucleic acid sequence of an intracellular domain of a co-stimulatory molecule and substantially lacks an extracellular ligand-binding domain. In one embodiment, the nucleic acid sequence encoding the chimeric intracellular signaling molecule is electroporated into the cells. In another embodiment, a nucleic acid sequence encoding a bispecific antibody is further electroporated into the cells. In yet another embodiment, a nucleic acid sequence encoding a CAR is further electroporated into the cells.

[0224] Optionally, the present invention includes a method for metabolically enhancing tumor-specific T cells, comprising introducing a CAR into T cells, wherein the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule; equipping the CAR T cells with a bispecific antibody, wherein the bispecific antibody binds to a target on tumor cells and the CAR T cells; and stimulating at least one co-stimulatory molecule on the equipped CAR T cells, wherein stimulation activates the intracellular domain of the co-stimulatory molecule, thereby metabolically enhancing the equipped T cells. In one embodiment, introducing the CAR into the T cells includes introducing a nucleic acid sequence encoding the CAR, such as mRNA encoding the CAR via electroporation. In another embodiment, equipping the CAR T cells includes contacting the CAR T cells with a bispecific antibody. In yet another embodiment, equipping the CAR T cells includes introducing a nucleic acid sequence encoding a bispecific antibody, such as mRNA encoding a bispecific antibody via electroporation. In yet another embodiment, stimulating the equipped CAR T cells improves the cytotoxicity of the equipped CAR T cells and their resistance to immunosuppression when in the tumor microenvironment. In yet another embodiment, the method further includes irradiating CAR T cells with up to 2500 rads to inhibit CAR T cell proliferation without inhibiting cytokine secretion or inducing cytotoxicity.

[0225] Sources of T cells Metabolically enhanced T cells can be generated from T cells of any source. In one embodiment, the source of T cells is obtained from an object. Non-limiting examples of objects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the object is a human. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In some embodiments, any number of T cell lines available in the art can be used. In some embodiments, any number of techniques known to those skilled in the art, such as Ficoll, can be used to separate T cells from a unit of blood collected from the object. In one embodiment, cells from circulating blood from an individual are obtained by apheresis or leukocyte extraction. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected via apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps, such as phosphate-buffered saline (PBS) or a washing buffer, which is calcium-deficient and may be magnesium-deficient or significantly lacking divalent cations. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca-free and Mg-free PBS. Alternatively, unwanted components in the apheresis sample can be removed, and the cells can be resuspended directly in a culture medium.

[0226] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation via a PERCOLL™ gradient. Optionally, T cells can be isolated from the umbilical cord. In any case, specific subsets of T cells can be further isolated using positive or negative selection techniques.

[0227] The umbilical cord blood mononuclear cells thus isolated can be depleted of cells expressing certain antigens—including but not limited to CD34, CD8, CD14, CD19, and CD56. This depletion can be accomplished using isolated antibodies, biological samples including ascites fluid containing antibodies, antibodies bound to a physical support, and cell-bound antibodies.

[0228] Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies targeting cell surface markers unique to negatively selected cells. Preferred methods include cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies targeting cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a mixture of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0229] To separate the desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a concentration greater than 100 million cells / ml is used. In a further embodiment, concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 × 10⁻⁶ are used. 6 Cell concentration of cells / ml. In yet another embodiment, 75, 80, 85, 90, 95, or 100 × 10⁶ cells / ml are used. 6 A cell concentration of cells / ml. In a further embodiment, 125 or 150 × 10⁻⁶ cells / ml can be used. 6 A concentration of cells / ml. Using high concentrations can produce increased cell yield, cell activation, and cell expansion.

[0230] Following the washing step, T cells can also be frozen, which does not require a monocyte removal step. While not wishing to be bound by theory, freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and a certain degree of monocytes from the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a cryogenic solution. While many cryogenic solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1° / min and stored in the vapor phase of a liquid nitrogen tank. Other methods of controlled freezing can be used, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen.

[0231] In one embodiment, the cell population includes the T cells of the present invention. Examples of cell populations include, but are not limited to, peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells include a T cell population. In yet another embodiment, purified T cells include a T cell population.

[0232] T cell expansion T cells generated by any of the methods described herein can be expanded in vitro. In one embodiment, T cells or a cell population comprising T cells are expanded. Typically, in the presence or absence of IL-2, T cells are expanded by contact with a surface having an agent attached thereto that stimulates CD3 / TCR complex-related signals and ligands that stimulate co-stimulatory molecules on the surface of the T cells.

[0233] This document describes methods for expanding T cells, whereby T cells can be expanded by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 10,000,000, or more times, and any and all whole or partial integers therebetween. In one embodiment, T cells are expanded in the range of approximately 20 to approximately 50 times.

[0234] Before passage the cells to another culture device, T cells can be cultured in cell culture medium in the culture device for a period of time or until the cells reach a confluence or high cell density for optimal passage. The culture device can be any culture device generally used for in vitro cell culture. Preferably, the confluence level is 70% or greater before passage to another culture device. More preferably, the confluence level is 90% or greater. The period of time can be any time suitable for in vitro cell culture. The T cell culture medium can be replaced at any time during T cell culture. Preferably, the T cell culture medium is replaced approximately every 2 to 3 days. The T cells are then harvested from the culture device, so that the T cells can be used immediately or refrigerated for later use. In one embodiment, the invention includes refrigerated expanded T cells. The refrigerated T cells are thawed before the introduction of one or more of the molecules described elsewhere herein into the T cells.

[0235] The culture steps described herein (contact with reagents, as described herein) can be very short, for example, less than 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture steps described further herein (contact with reagents, as described herein) can be longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0236] In one embodiment, T cells can be cultured for an integer value of several hours (approximately 3 hours) to approximately 14 days or any hour in between. Suitable conditions for T cell culture include appropriate culture media (e.g., minimally essential medium or RPMI 1640 or X-vivo 15, (Lonza)) that may contain factors essential for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma protein powder, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, an Optimizer (with added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined hormone set), and / or sufficient amounts of cytokines (one or more) for the growth and expansion of T cells. Antibiotics such as penicillin and streptomycin are included only in the experimental culture and not in the cell culture of the target cells. Target cells are maintained under conditions necessary for growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0237] T cell culture media may include reagents that can co-stimulate T cells. For example, a reagent that can stimulate CD3 is an antibody against CD3, and a reagent that can stimulate CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can be expanded by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000, or more. In one implementation, T cells are expanded by culturing electroporation populations to a range of approximately 20 to approximately 50 times or more.

[0238] therapy The metabolically enhanced T cells described herein are useful in various therapeutic modalities for treating a wide range of disease states, regardless of whether the T cells are metabolically enhanced by expressing either a chimeric intracellular signaling molecule or a CAR. Therefore, regardless of whether the T cells express a chimeric intracellular signaling molecule or a CAR, the T cells are referred to herein as metabolically enhanced T cells. Compositions comprising metabolically enhanced T cells can be prepared according to methods described elsewhere herein. These metabolically enhanced T cells can be included in the composition for use in the therapies as now described.

[0239] In one aspect, the composition comprises metabolically enhanced T cells that include the chimeric intracellular signaling molecules described herein. In another aspect, the composition comprises metabolically enhanced cells that further include the bispecific antibody described herein. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells can be administered.

[0240] In one aspect, the present invention includes a method of treating a disease or symptom in a subject associated with enhanced immunity, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In another aspect, the present invention includes a method of treating a symptom in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In yet another aspect, the present invention includes a method for stimulating a T cell-mediated immune response against target cells or tissues in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In yet another aspect, the present invention includes the use of the metabolically enhanced T cells as described herein in the manufacture of medicaments for treating an immune response in a subject in which they are desired. In these embodiments, the T cells comprise chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules comprise an intracellular domain of a co-stimulatory molecule and substantially lack an extracellular ligand-binding domain. In another embodiment, the T cells further comprise bispecific antibodies. In yet another embodiment, the T cells further comprise CARs.

[0241] In one aspect, the present invention includes a method of treating a disease or condition associated with a tumor or cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In another aspect, the present invention includes a method of treating a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In yet another aspect, the present invention includes a method for stimulating a T cell-mediated immune response against target tumor cells or tumor tissue in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising metabolically enhanced T cells as described herein. In yet another aspect, the present invention includes the use of the metabolically enhanced T cells as described herein in the manufacture of a medicament for treating a tumor or cancer in a subject in which they are desired. In these embodiments, the T cells comprise a CAR and a bispecific antibody, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule, and the bispecific antibody binds to a target on tumor cells and the T cells.

[0242] The metabolically enhanced T cells described herein can be administered to animals, preferably mammals, and even more preferably humans, to suppress immune responses, such as those common to autoimmune diseases like diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhancing allograft tolerance induction, and transplant rejection. Furthermore, the metabolically enhanced T cells of the present invention can be used to treat any condition in which a weakened or otherwise suppressed immune response, particularly a cell-mediated immune response, is desired to treat or alleviate the disease. In one aspect, the invention includes treating conditions such as autoimmune diseases, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the cell population described herein.

[0243] Examples of autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Bechtel's disease, cardiomyopathy, celiac-herpetic dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos' disease, juvenile dermatomyositis, discoid lupus, idiopathic mixed cryoprecipitate globulinemia, fibromyalgia-fibromyositis, Graves' disease, Graves-Bartholin's syndrome, and Hashimoto's dermatitis. Thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Stiele's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, nodular hyperkinesia, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia. Primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Leter syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff body syndrome, systemic lupus erythematosus, Angoni's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, leukoplakia, and Wegener's granulomatosis.

[0244] The metabolically enhanced T cells described in this article can also be used to treat inflammatory disorders. Examples of inflammatory disorders include, but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, specific allergies, allergic reactions, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-induced lung injury.

[0245] The metabolically enhanced T cells of this invention can be used to treat cancer. Cancer includes tumors that are not vascularized or have not yet been significantly vascularized, as well as vascularized tumors. Cancer can include non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) or can include solid tumors. Types of cancer for which cell therapy of this invention is intended include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0246] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-derived) cancers include leukemia, which includes acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0247] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, mesotheliomas, Ewing's tumors, leiomyosarcoma, rhabdomyosarcoma, colon cancer, malignant lymphomas, pancreatic cancer, breast cancer, lung cancer, ovarian 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, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, and bile duct carcinoma. Choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neurocytoma, retinoblastoma, and brain metastases).

[0248] The metabolically enhanced T cells of the present invention can be administered at various doses and via various routes, and are sometimes determined in appropriate preclinical and clinical trials and experiments. The cell composition can be administered multiple times at doses within these ranges. The administration of the metabolically enhanced T cells of the present invention can be combined with other methods, as determined by those skilled in the art, that are useful for treating the desired disease or condition.

[0249] The metabolically enhanced T cells of the present invention can be applied to subjects who have undergone therapy, whether they are autologous, allogeneic, or xenogeneic.

[0250] The administration of the metabolically enhanced T cells of the present invention can be carried out in any conventional manner known to those skilled in the art. The metabolically enhanced T cells of the present invention can be administered to the subject via nebulization, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered via arteries, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intravenously. iv It can be injected or administered intraperitoneally to the patient. In other cases, the metabolically enhanced T cells of the present invention are directly injected into sites of inflammation, localized disease sites, lymph nodes, organs, tumors, etc. in the subject.

[0251] Pharmaceutical Composition The pharmaceutical compositions of the present invention may include metabolically enhanced T cells as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0252] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of the disease. The dosage and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined through clinical trials.

[0253] When the instructions specify "immunely effective dose," "anti-immune response effective dose," "immune response-suppressive effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, immune response, and patient (subject) condition. Typically, it can be specified that the dosage can be in units of 10... 4 Up to 10 9 Cells / kg body weight, preferably 10 5 Up to 10 6The pharmaceutical composition comprising T cells described herein is administered at a dose of cells per kg body weight—including all integer values ​​within those ranges. The T-cell composition may also be administered multiple times at these doses. The cells can be administered using infusion techniques 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 specific patient can be readily determined by a person skilled in the medical field by monitoring the patient’s signs of disease and adjusting treatment accordingly.

[0254] In some embodiments, it may be desirable to administer metabolically enhanced T cells to the subject, and subsequently redraw blood (or perform apheresis) to metabolically enhance the T cells derived therefrom according to the invention, and then reinfuse the patient with these metabolically enhanced T cells. This process may be performed multiple times every few weeks. In some embodiments, metabolically enhanced T cells can be obtained from blood draws ranging from approximately 10 ml to approximately 400 ml. In some embodiments, metabolically enhanced T cells are obtained from blood draws of approximately 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100 ml. Without being bound by theory, certain T cell populations can be selected using this multiple blood draw / reinfusion protocol.

[0255] In some embodiments of the invention, T cells are metabolically enhanced using the methods described herein, and T cells are stimulated, activated, or expanded using the methods described herein or other methods known in the art, wherein the T cells are expanded to a therapeutic level and are administered to the patient along with any number of relevant treatment modalities (e.g., before, during, or after), including but not limited to treatments using pharmaceutical agents such as antiviral therapy for MS patients or PML patients, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab. In a further embodiment, the metabolically enhanced T cells of the present invention can be used in combination with: chemotherapy, radiation, 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, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines, and irradiation. These drugs inhibit calcium-dependent phosphatases (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling. (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). In a further embodiment, the metabolically enhanced T-cell composition of the present invention is administered to the patient along with bone marrow transplantation, using a chemotherapy agent such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH for T-cell elimination therapy (e.g., before, simultaneously with, or after). In another embodiment, the cell composition of the present invention is administered after B-cell elimination therapy—such as an agent that reacts with CD20, such as Rituxan. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In another implementation, the expanded cells are applied before or after surgery.

[0256] The dosage of the above treatment administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be determined according to practice accepted in the art. For example, the dosage of CAMPATH for adult patients will typically be in the range of 1 to about 100 mg, often administered daily for a period between 1 and 30 days. While larger doses up to 40 mg / day may be used in some cases (described in U.S. Patent No. 6,120,766), the preferred daily dose is 1 to 10 mg / day.

[0257] It should be understood that the methods and compositions useful in this invention are not limited to the specific formulations set forth in the examples. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to manufacture and use cells, methods for expansion and culture, and treatments of the present invention, and are not intended to limit the scope of the invention as perceived by the inventors.

[0258] Unless otherwise indicated, the practice of this invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the knowledge of those skilled in the art. Such techniques are well described in, for example, "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook et al., (2012) Molecular Cloning, Cold Spring Harbor Laboratory); "Oligonucleotide Synthesis" (Gait, MJ (1984). Oligonucleotide synthesis. IRL press); "Culture of Animal Cells" (Freshney, R. (2010). Culture of animalcells. Cell proliferation, 15(2.3), 1); "Methods in Enzymology" "Weir'sHandbook of Experimental Immunology" (Wiley-Blackwell; 5 edition (January15, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Carlos, (1987) Cold Spring Harbor Laboratory, New York); "Short Protocols in Molecular Biology" (Ausubel, et al. al.,Current Protocols; 5 edition, November 5, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, M., VDM Verlag Dr. Müller, August 17, 2011); “Current Protocols in Immunology” (Coligan, John Wiley & Sons, Inc., November 1, 2002). These techniques are applicable to the production of the polynucleotides and peptides of the present invention, and therefore can be considered for the purpose of completing and practicing the present invention.Particularly useful techniques for specific implementations will be discussed in the following sections.

[0259] Experimental Examples The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise specified, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent from the teachings provided herein.

[0260] Without further description, it is believed that those skilled in the art can make and utilize the compounds of the present invention and practice the claimed methods using the prior description and the following illustrative examples. Therefore, the following working examples specifically point to preferred embodiments of the invention and are not to be construed as limiting the remainder of this disclosure in any way.

[0261] The materials and methods used in these experiments are now described.

[0262] Bispecific antibodies are generated. Bispecific antibodies (BiAbs) are generated through chemical heteroconjugation as described above with Omega-3 and Rituximab (humanized anti-CD20 IgG1, Genentech Inc., South San Francisco, CA), Omega-3 and Erbitux (humanized anti-epidermal growth factor receptor (EGFR) IgG1, ImClone LLC., Branchburg, NJ), or Omega-3 and Herceptin (humanized anti-HER2 IgG1, Genentech Inc., South San Francisco, CA). (Gall JM, et al.) Experimental Hematology 2005, 33:452-459; Reusch U, et al., Journal of Hematotherapy and Stem Cell Research 2001, 10:247-260).

[0263] Equip T cells. Use the previously optimized concentration (50 ng / 10⁻⁶). 6 ATC), equipped activated T cells with anti-OKT3 x anti-CD20 BiAb (CD20Bi) or anti-OKT3 x anti-EGFR BiAb (EGFRBi) (Sen M, et al., Journal of Hematotherapy and Stem Cell Research 2001, 10:247-260).

[0264] Cytotoxicity / 51Cr release assay. To target adherent cells, 4 × 10⁻⁶ Cr was released. 4 Cells were seeded at 100 cells / well in 96-well flat-bottom microtiter plates and allowed to adhere overnight. Cells were then inoculated in labeled medium (50% FBS in complete RPMI-1640) at 37°C at 20 µCi / mL. 51 Cells were Cr-labeled for 5 hours and washed with full RPMI-1640 to remove unincorporated isotopes. For non-adhesive cell targeting, cells were incubated in 15 mL conical tubes at 37°C with 100 µCi / 10⁻¹⁰. 6 Use per cell 51 Cr-labeled Daudi cells for 2 hours, washed with complete RPMI-1640, and then... 4 Cells / well plates were seeded in 96-well round-bottom microtiter plates. Effector (unequipped CART19 or ATC and equipped CART19 or ATC) were then added to achieve a 10:1 effector:target (E:T) ratio. Co-cultures were incubated for 4 hours (Daudi) or 18 hours (adhesive cell lines), and the supernatant was collected for liquid scintillation counting to quantify the release. 51 The amount of Cr. Calculate percentage cytotoxicity as follows: (experimental cpm - spontaneous cpm) / (maximum cpm - spontaneous cpm) × 100. Calculate the mean and standard error from four to six replicates / samples.

[0265] The results of the experiment will now be described.

[0266] When exposed to hypoxic conditions, CAR-modified T cells exhibited intracellular signaling and surprising survival. Figure 1 This is a set of graphs showing the survival of CAR cells under hypoxic conditions. Survival varies depending on the type of CAR structure expressed in T cells. Survival differences depend on co-stimulatory intracellular domains. Figure 2 The study showed that different co-stimulatory intracellular domains were tested in the hippocampus assay to influence the metabolic activity of CAR-expressing T cells.

[0267] Figure 3 The study demonstrated the metabolic activity of T cells expressing CAR-co-stimulated intracellular domains of CD28z or 4-1BBz. 4-1BBz CAR T cells exhibit ligand-independent signaling and are reprogrammed as central memory T (Tcm) cells, while CD28z CAR T cells are reprogrammed as effector memory (Tem) cells. 4-1BBz CAR T cells have lower levels of surface molecules such as PD1, TIM3, and LAG3, which are typically associated with depletion. They also exhibit higher metabolic activity upon activation by alternative antigens and survive longer under strictly hypoxic conditions. Figure 3 ).

[0268] Using CARs has shown metabolically enhanced modified T cells and may reduce the alloreactivity of modified T cells. Such findings support the use of "universal T cells" or even third-party T cells. CARs can be introduced into T cells via various methods, such as using lentiviral vectors (…). Figure 4 or mRNA electroporation ( Figure 5 Potential targets for CARs include any tumor-associated antigens, such as EGFR, HER2 / NEU, PSMA, BCMA, GD2, mesothelin, etc. The intracellular portion of the CAR used in the trials described herein may include one or more signaling domains selected to enhance T cell metabolism against any given tumor microenvironment, such as 4-1BB, CD27, 0x40, ICOS, or CD28. Exposing bispecific scFvs to antibodies or "equipping" the CAR cells immediately prior to infusion into the patient, or covalently conjugating bispecific antibodies to CAR cells, can also be useful for enhancing T cell cytolytic activity.

[0269] As an alternative to using lentiviral vectors, short-lived T cells can be metabolically enhanced by introducing CAR-encoding mRNA via electroporation. Figure 5 In short, T cells are cultured and co-stimulated with anti-CD3 and anti-CD28 antibodies, with or without IL-2. The T cells are then electroporated with in vitro transcribed mRNA to introduce CARs. The CAR cells are then equipped with bispecific antibodies. Optionally, in vitro transcribed mRNA encoding a bispecific scFv or a bispecific antibody can be electroporated into the CAR cells immediately prior to infusion. The bispecific antibody-equipped CAR cells can then be injected intravenously or intratumorally into the patient.

[0270] Figure 6 The figures show the cytotoxicity of equipped (with bispecific antibodies) and unequipped (without bispecific antibodies) CART cells versus non-CART cells. Effector cells include unequipped CART19 cells and CART19 cells equipped with anti-HER2, anti-EGFR, or anti-CD20 bispecific antibodies (top figure), as well as unequipped non-CART cells and non-CART cells equipped with anti-HER2, anti-EGFR, or anti-CD20 bispecific antibodies (bottom figure). Both CART19 cells and non-CART cells exhibited cytotoxicity against target cells.

[0271] The cytotoxicity ratio of unequipped and equipped CART19 cells was measured during repeated exposure to SKBR3 on days 1, 4, 10, 18, and 25. Figure 7The left figure shows a high level of cytotoxicity against the target cells, even after multiple rounds of the killing assay (in the absence of IL-2). Figure 7 The right panel shows the enhanced proliferation exhibited by CART19 cells during repeated kill experiments.

[0272] Figures 8A-8E The study demonstrated the cytotoxicity of equipped or unequipped CAR19 cells and non-CAR T cells against various target cells expressing HER2 or EGFR. Effector cells were equipped with anti-HER2 or anti-EGFR bispecific antibodies. CAR19 cells equipped with anti-HER2 bispecific antibodies were effective in killing even low-HER2-expressing (MCF-7) or non-HER2-expressing breast cancer cell lines (triple-negative [HER2 / ER / PR-negative cell lines] MB231 and BT-20), with an efficacy of ~20-50%.

[0273] Figure 9 CART19 cells equipped with (HER2 bispecific antibody) showed increased expression of both activating and inhibitory co-receptors, such as 4-1BB, ICOS, OX40, and PD1 on CD4 and CD8 T cells.

[0274] Intracellular signaling domains derived from other co-stimulatory molecules can also play a positive role in metabolic activity in T cells. Chimeric intracellular signaling molecules ( Figure 10 The intracellular domain of SEQ ID NO:1 can be selected from co-stimulatory molecules that enhance T cell metabolism in response to any given tumor microenvironment, such as 4-1BB, CD27, Ox40, ICOS, or CD28.

[0275] The anticipated chimeric intracellular signaling molecules include detectable markers for surface detection. Figure 10 Otherwise, it cannot be detected on the surface of T cells. Alternatively, alternative markers can be used in place of detectable markers, such as fluorescent proteins or cell surface molecules co-expressed by the T2A or IRES system. Figure 11 Detectable markers can also be used in conjunction with alternative markers. Figure 12 ).

[0276] Chimeric intracellular signaling molecules can also be transmitted via lentiviral vectors ( Figure 13 or mRNA electroporation ( Figure 14 Chimeric T cells are introduced into T cells. Similar to CAR-T cells, by loading or equipping them with proteins just before infusion into the patient, these chimeric T cells modified with intracellular signaling molecules can exhibit bispecific scFv or be conjugated with bispecific antibodies.

[0277] Chimeric intracellular signaling molecules can also be introduced into T cells via mRNA electroporation. Figure 14 The in vitro transcribed mRNA is electroporated into T cells before being infused into the patient to equip the T cells with bispecific scFv or bispecific antibodies.

[0278] It is hypothesized that T cells equipped with bispecific antibodies (BiAb) exhibit enhanced cytotoxicity against target cells. Figure 15 This diagram illustrates the mechanism of equipping T cells with bispecific antibodies—produced through a chemical heteroconjugation of anti-CD3 (mouse monoclonal antibody-CD3, OKT3)—and the specificity of these bispecific antibodies against any selected antigen (tumor, virus, bacteria, or parasite). Bispecific antibody-equipped T cells should be reactivated to exhibit non-MHC-restricted cytotoxicity against target cells. The bispecific antibody-equipped T cells then produce cytokines and chemokines after binding with target cells.

[0279] When bound to tumor targets, irradiation of T cells equipped with bispecific antibodies blocks the allogeneic response. Figure 16 (Left image) but does not inhibit the cytotoxicity of equipped T cells ( Figure 16 (See the right image). "r" represents effector cells, and "s" represents stimulator cells. Indicates the irradiated cells.

[0280] Figure 17 The study demonstrated metabolically enhanced T cells, whether unequipped or equipped with anti-CD20 bispecific antibody (CD20Bi), anti-EGFR bispecific antibody (EGFRBi), anti-GD2 bispecific antibody (GD2Bi), and anti-HER2 bispecific antibody (HER2Bi), against breast cancer and pancreatic / colorectal cancer cell lines expressing HER2 and EGFR. 51 Cr release assay for cytotoxicity. Cytotoxicity was measured using equipped or unequipped metabolically enhanced T cells in triplicate for 18 hours at an effector / target ratio of 10:1.

[0281] In the absence of a target cell or in the presence of target cells at an E:T ratio of 10:1 (SKBR3), unequipped or equipped T cells or non-metabolic enhanced ATCs were seeded overnight. The presence of cytokines in the culture supernatant was determined using a Bio-Plex assay. Figure 18 (as shown in the above figure) and chemokines ( Figure 18 (See the image below). The value was reported as pg / ml.

[0282] To determine whether chimeric intracellular signaling molecules express metabolically enhanced T cells, normal donor ND317 T cells were transduced with 160 µl or 400 µl of virus to express tagged chimeric intracellular signaling molecules, including the CD8 transmembrane domain and the 4-1BB and CD3ζ intracellular domains. This transduction resulted in a slightly slower rate of expansion (a slower decrease in cell size and doubling). Figure 19A ). Metabolically enhanced T cells that enrich expression of chimeric intracellular signaling molecules labeled with tags on CD4+ cells ( Figure 19B The percentage of T cells expressing tag-labeled chimeric intracellular signaling molecules decreased from day 6 to day 8 in the total population. Figure 19C ).

[0283] Figure 19C The image shows an expanded profile of T cells expressing tag-tagged chimeric intracellular signaling molecules—CD8 transmembrane domains with 4-1BB and CD3ζ intracellular domains or membrane-anchored myristylated 4-1BB and CD3ζ intracellular domains.

[0284] Figure 20 The image shows normal donor ND444 T cells transduced with high (50%) and low (15%) viral loads. Time-tagged T cells were lost upon detection. However, metabolically enhanced T cells enriched for CD4+ cells (tag+).

[0285] Comparison of expansion of normal donor ND422 T cells between cells labeled with transduction tag-tagged chimeric intracellular signaling molecules (BBz) or CAR19BBz. Figure 21A A slightly slower population doubling rate and reduced cell size were observed in cells transduced with the tag-BBz. The percentage of CD4 background expression was high for this donor, as was observed in untransduced cells on day 5. A slight decrease in the percentage of CD4 population was observed on day 8. Figure 21B However, this donor showed no decrease in the percentage of tagged T cells observed from day 5 to day 8. Figure 21C ).

[0286] Other implementation methods The description of the list of elements in any definition of a variable in this document includes the definition of the variable as any single element or a combination (or sub-combination) of the listed elements. The description of an implementation in this document includes the implementation as any single implementation or in combination with any other implementation or parts thereof.

[0287] The disclosure of each and every patent, patent application, and publication cited herein is hereby incorporated in its entirety by reference. While the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

Claims

1. Metabolically enhanced T cells, comprising chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules include: At least one intracellular domain of a cardamomylation signaling and co-stimulatory molecule; The chimeric intracellular signaling molecules described therein are essentially lacking extracellular domains and ligand-binding domains; The chimeric intracellular signaling molecules mentioned above do not include hinge domains or transmembrane domains. The co-stimulatory molecules mentioned therein are selected from T cell receptor (TCR), CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3; The T cells express the chimeric intracellular signaling molecules and stimulate at least one co-stimulatory molecule on the T cells to activate the chimeric intracellular signaling molecules, thereby metabolically enhancing the T cells; and The activation of the chimeric intracellular signaling molecules improves T cell toxicity and resistance to immunosuppression in the tumor microenvironment.

2. Metabolically enhanced T cells, comprising chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules include: The hinge domain, the transmembrane domain, and at least one intracellular domain of the co-stimulatory molecule; The chimeric intracellular signaling molecules described therein are essentially lacking extracellular domains and ligand-binding domains; The co-stimulatory molecules mentioned therein are selected from T cell receptor (TCR), CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3; The hinge domain and / or the transmembrane domain are selected from the following: α chain of TCR, β chain of TCR, ζ chain of TCR, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIR, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R β, IL2R γ, IL7R. α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF1, SLAMF8, SLAMF6, SELPLG Hinge domains and / or transmembrane domains of molecules such as (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or any combination thereof. The T cells express the chimeric intracellular signaling molecules and stimulate at least one co-stimulatory molecule on the T cells to activate the chimeric intracellular signaling molecules, thereby metabolically enhancing the T cells; and The activation of the chimeric intracellular signaling molecules improves T cell toxicity and resistance to immunosuppression in the tumor microenvironment.

3. Metabolically enhanced T cells, comprising chimeric intracellular signaling molecules, wherein the chimeric intracellular signaling molecules include: The intracellular domain of the co-stimulatory molecule and the non-antigen-binding extracellular domain of the Fc, including IgD or IgA. The co-stimulatory molecules mentioned therein are selected from T cell receptor (TCR), CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3; The T cells express the chimeric intracellular signaling molecules and stimulate at least one co-stimulatory molecule on the T cells to activate the chimeric intracellular signaling molecules, thereby metabolically enhancing the T cells; and The activation of the chimeric intracellular signaling molecules improves T cell toxicity and resistance to immunosuppression in the tumor microenvironment.

4. The metabolically enhanced T cells according to any one of claims 1-3, wherein the chimeric intracellular signaling molecules further comprise detectable markers.

5. The metabolically enhanced T cells according to any one of claims 1-3, further comprising a bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that binds to a target cell and a second antigen-binding domain that binds to an activated T cell.

6. A pharmaceutical composition comprising a metabolically enhanced T cell and a pharmaceutically acceptable carrier according to any one of claims 1-3.

7. Use of the metabolically enhanced T cells according to any one of claims 1-3 in the manufacture of a medicament for treating an immune response in a subject in need of it.

8. The use according to claim 7, wherein the treatment comprises administering a therapeutically effective amount of the drug to the subject.

9. The metabolically enhanced T cell of claim 2, wherein the hinge domain is a CD8-α hinge domain and the transmembrane domain is a CD28 transmembrane domain.

10. The metabolically enhanced T cell of claim 3, further comprising a bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain specifically binding to the Fc of the IgD or IgA and a second antigen-binding domain binding to the target antigen or T cell antigen.

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