CN116286665A - Chimeric antigen receptor cells secrete therapeutic agents - Google Patents

Abstract

The present disclosure relates to compositions and methods for enhancing T cell responses. Some embodiments relate to a cell comprising an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding at least one of the therapeutic agents IL-12, IL-2, IL-6, IL-7, IL-15, IL-17, ifn- γ, and IL-23 as or comprising. The cells express and secrete the therapeutic agent.

Description

Chimeric antigen receptor cells secrete therapeutic agents

The patent application of the invention aims at the application number: 201910542560.2, the filing date of the original application is: 2019-06-21, the invention is named: chimeric antigen receptor cells secrete therapeutic agents.

Technical Field

The present disclosure relates to compositions and methods relating to secretion of therapeutic agents by chimeric antigen receptor cells, and their use in the treatment of diseases including cancer.

Background

Cancer is known as a malignancy that involves abnormal cell growth and may invade or spread to other parts of the body. In humans, there are more than one hundred types of cancer. One example is breast cancer that occurs in breast epithelial tissue. Because breast cancer cells lose the characteristics of normal cells, the link between breast cancer cells is lost. Once cancer cells shed, they spread throughout the body through the blood and/or lymphatic system, thus being life threatening. Currently, breast cancer has become one of the common threats to female physical and mental health. Although immunotherapy (e.g., CAR T) has proven effective for treating cancer, there remains a need for improved immunotherapy to make it more effective against certain cancers such as solid tumors.

Disclosure of Invention

The technical scheme adopted by the invention is as follows:

embodiments relate to compositions and methods for enhancing T cell responses. Some embodiments relate to a cell comprising an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding a polypeptide comprising at least one of the therapeutic agents IL-12, IL-2, IL-6, IL-7, IL-15, IL-17, ifn- γ, and IL-23. The cells express and secrete the therapeutic agent.

This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For purposes of this disclosure, the following terms are defined as follows.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

By "about" is meant that the number, level, value, number, frequency, percentage, size, quantity, weight, or length varies by up to 20,15,10,9,8,7,6,4,3,2 or 1% to a reference number, level, value, quantity, frequency, percentage, size, quantity, weight, or length.

As used herein, the term "activation" refers to the state of a cell that has been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" particularly refers to a T cell undergoing cell division.

The term "antibody" is used in its broadest sense and refers to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. Antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, fv, fab and F (ab) 2, and single chain 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).

The term "antibody fragment" refers to a portion of a full length antibody, such as the antigen binding or variable regions of an antibody. Other examples of antibody fragments include Fab, fab ', F (ab') 2, and Fv fragments; a double body; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

The term "Fv" refers to the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain that are tightly, non-covalently, bound. Six hypervariable loops (3 loops from each of the H and L chains) emanate from the fold of these two domains, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three Complementarity Determining Regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site (dimer).

As used herein, an "antibody heavy chain" refers to the larger of two types of polypeptide chains that are present in a naturally occurring conformation in all antibody molecules. As used herein, an "antibody light chain" refers to the smaller of the two types of polypeptide chains that are present in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to two major antibody light chain isotypes.

The term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by phage. The term also includes antibodies produced by synthesizing DNA molecules encoding the antibodies and expression of the DNA molecules to obtain the antibodies or to obtain amino acids encoding the antibodies. Synthetic DNA is obtained using techniques available and well known in the art.

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

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

The term "self-antigen" refers to an antigen that is incorrectly recognized as foreign by the immune system. Autoantigens include cell proteins, phosphoproteins, cell surface proteins, cell lipids, nucleic acids, glycoproteins, including cell surface receptors.

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

The term "allograft" is used to describe grafts derived from different subjects of the same species. As an example, the donor subject may be a related or unrelated or recipient subject, but the donor subject has an immune system signature similar to the recipient subject.

The term "xenogeneic" is used to describe grafts derived from subjects of different species. For example, a donor subject is from a different species than a recipient subject, and the donor subject and recipient subject may be genetically and immunologically incompatible.

The term "cancer" is used to refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system. Examples of the various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprising," and "include" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The phrase "consisting of" is meant to include and be limited to anything after the phrase "consisting of. Thus, the phrase "consisting of" means that the listed elements are necessary or mandatory and that no other elements are present.

The phrase "consisting essentially of" is meant to include any element listed after the phrase, and may include other elements that do not interfere with or contribute to the activities or actions specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present, depending on whether they affect the activity or effect of the listed elements.

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

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

The term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by the binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, which mediates T cell responses including at least one of proliferation, activation, differentiation and others. Co-stimulatory ligands may include B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2,4-1BBL, OX40L, an inducible co-stimulatory ligand (ICOS-L), an intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, ligands for CD7, agonists or antibodies that bind Toll ligand receptors and ligands that specifically bind to B7-H3. Costimulatory ligands include, inter alia, agonists or antibodies that specifically bind to costimulatory molecules present on T cells, such as CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind CD 83.

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

The term "costimulatory signal" refers to a signal that, in combination with a primary signal (e.g., TCR/CD3 linkage), results in up-or down-regulation of T cell proliferation and/or a key molecule. The terms "disease" and "disorder" may be used interchangeably or may be different in that a particular disease or disorder may not have a known causative agent (and therefore the cause has not been resolved) and thus has not been recognized as a disease, but merely as an adverse condition or syndrome, wherein a clinician has established a more or less specific set of symptoms. The term "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of health in which an animal is able to maintain homeostasis, but in which the animal's state of health is less than in the absence of the disorder. If not treated in time, the disease does not necessarily lead to a further decline in the health of the animal.

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

The term "coding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for the synthesis of other polymers and macromolecules in biological processes, with any defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or defined amino acid sequence and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand, which has the same nucleotide sequence as the mRNA sequence (except that "T" is replaced by "U") and is typically provided in the sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as a protein or other product encoding the gene or cDNA.

The term "exogenous" refers to a molecule that does not occur naturally in a wild-type cell or organism but is typically introduced into the cell by molecular biology techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or artificial nucleic acid constructs encoding the desired protein. With respect to polynucleotides and proteins, the term "endogenous" or "native" refers to naturally occurring polynucleotide or amino acid sequences that can be found in a given wild-type cell or organism. Furthermore, a particular polynucleotide sequence that is isolated from a first organism and transferred to a second organism by molecular biological techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In particular embodiments, polynucleotide sequences may be "introduced" by molecular biological techniques into microorganisms that already contain such polynucleotide sequences, for example, to produce one or more additional copies of additional naturally occurring polynucleotide sequences, and thereby facilitate overexpression of the encoded polypeptide.

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

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

The term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when positions in two compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if positions in each of the two polypeptides. The DNA molecule is occupied by adenine and then the molecule is homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, if 6 of the 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. The comparison is made when the two sequences are aligned to produce maximum homology.

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

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

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

In the context of the present disclosure, the following abbreviations for the ubiquitous nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding a protein may comprise the intron(s) in some versions.

The term "lentivirus" refers to a genus of the retrovirus family. Lentiviruses are unique among retroviruses capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, and therefore they are one of the most effective methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

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

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA of the pre-sequence or secretion leader is expressed as a pre-protein involved in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned for translation.

The term "under transcriptional control" refers to a promoter operably linked to a polynucleotide and in the correct position and orientation to control the initiation of transcription of an RNA polymerase and the expression of the polynucleotide.

The term "over-expressed" tumor antigen or "overexpression" of a tumor antigen is intended to mean an abnormal expression level of the tumor antigen in cells from a disease region, such as a solid tumor within a particular tissue or organ associated with a patient, into normal cells from that tissue or organ. Patients characterized by solid tumors or hematological malignancies with overexpression of tumor antigens can be determined by standard assays known in the art.

Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors may be benign or malignant. Different types of solid tumors are named for the cell types that they form (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid medullary carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, nephroblastoma, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and central nervous system tumors (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, central nervous system lymphoma, germ cell tumor, medulloblastoma, schwannoma, neuroblastoma, angioma, angioblastoma, and retinoblastoma.

The solid tumor antigen is an antigen expressed on a solid tumor. In embodiments, the solid tumor antigen is also expressed at low levels on healthy tissue.

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

The terms "patient," "subject," and "individual" and the like are used interchangeably herein and refer to any person, animal, or organism suitable for use in the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human or animal. In embodiments, the term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans and animals such as dogs, cats, mice, rats and transgenic species thereof.

A subject in need of treatment or in need thereof includes a subject suffering from a disease, disorder or condition in need of treatment. Subjects in need thereof also include subjects in need of treatment to prevent a disease, disorder, or condition.

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

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

The terms "polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. In certain aspects, a polypeptide may include an enzymatic polypeptide or "enzyme" that generally catalyzes (i.e., increases the rate of) various chemical reactions.

The term "polypeptide variant" refers to a polypeptide that is distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, the polypeptide variants are distinguished from the reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, polypeptide variants comprise conservative substitutions, and in this regard, it is well known in the art that certain amino acids may be changed to amino acids having broadly similar properties without altering the nature of the polypeptide activity. Polypeptide variants also include polypeptides in which one or more amino acids have been added or deleted or replaced with a different amino acid residue.

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

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

By "statistically significant" is meant that the result is unlikely to happen by chance. Statistical significance may be determined by any method known in the art. Common important metrics include the p-value, i.e., the frequency or probability that an observation event will occur if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined as a p value of 0.5 or less. The "reducing" or "reducing" amount is typically a "statistically significant" or physiologically significant amount and may include a reduction of about 1.1,1.2,1.3,1.4,1.5,1.6 1.7,1.8,1.9,2,2.5,3,3.5,4,4.5,5,6,7,8,9,10,15,20,30,40 or 50 times or more (e.g., 100,500,1000 times) (including 1 and all integers and fractions above 1, e.g., 1.5,1.6,1.7.1.8, etc., the amounts or levels described herein).

The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as the down regulation of TGF- β and/or recombination of cytoskeletal structures.

The term "stimulatory molecule" refers to a molecule that specifically binds to a T cell of a cognate stimulatory ligand present on an antigen presenting cell. For example, the functional signaling domain derived from a stimulatory molecule is a zeta chain associated with the T cell receptor complex.

The term "stimulatory ligand" refers to the expression on a cell (e.g., a T cell) of a ligand (referred to herein as a "stimulatory molecule") that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner, thereby mediating a primary response of the T cell, including activating, initiating an immune response, proliferating and the like. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD 3 antibodies, super agonist anti-CD 28 antibodies and super agonist anti-CD 2 antibodies.

The term "therapeutic agent" refers to treatment and/or prevention. Therapeutic effects are obtained by inhibiting, alleviating or eradicating a disease state or alleviating symptoms of a disease state.

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

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

The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. Cells include the primary test cells and their progeny.

The term "vector" refers to a polynucleotide that comprises an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term also includes non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like. For example, lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. Lentiviral vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: SIV. Lentiviral vectors are generated by attenuating HIV virulence genes multiple times, e.g., genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.

The range is as follows: throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have all possible subranges as specifically disclosed, as well as individual numerical values within that range. For example, descriptions of ranges such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual values within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

A "chimeric antigen receptor" (CAR) molecule is a recombinant polypeptide that includes at least an extracellular domain, a transmembrane domain, and a cytoplasmic domain or an intracellular domain. In embodiments, the domains of the CAR are located on the same polypeptide chain, e.g., a chimeric fusion protein. In embodiments, the domains are located on different polypeptide chains, e.g., the domains are not contiguous.

The extracellular domain of the CAR molecule includes an antigen binding domain. In embodiments, the antigen binding domain binds an antigen on the surface of a B cell, such as a cell surface molecule or a marker. In embodiments, the cell surface molecule of the B cell comprises CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11B, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cell surface molecule of the B cell is CD19, CD20, CD22 or BCMA. In a particular embodiment, the cell surface molecule of the B cell is CD19.

In embodiments, the antigen binding domain binds an antigen on the tumor surface, e.g., a tumor antigen or tumor marker. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly T cell mediated immune responses. Tumor antigens are well known in the art and include, for example, tumor-associated MUC1, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, her2/neu, survival, telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin. For example, when the tumor antigen is CD19, its CAR may be referred to as a CD19CAR.

In embodiments, the extracellular antigen-binding domain of the CAR comprises at least one scFv or at least one single domain antibody. For example, there may be two scfvs on the CAR. The scFv comprises a light chain variable region (VL) and a heavy chain variable region (VH) of a target antigen-specific monoclonal antibody linked by a flexible linker. Single chain variable region fragments can be prepared by ligating light and/or heavy chain variable regions using short connecting peptides (Bird et al, science 242:423-426, 1988). An example of a linker peptide is a GS linker having the amino acid sequence (GGGGS) 3 (SEQ ID NO: 20) bridging between the carboxy terminus of one variable region and the amino terminus of the other variable region by about 3.5nm. Other sequences of linkers have been designed and used (Bird et al, science 242:423-426,198). In general, the linker may be a short, flexible polypeptide, and preferably comprises about 20 or fewer amino acid residues. Single-chain variants may be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer may be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv may be introduced into a suitable host cell, eukaryotic cell, such as yeast, plant, insect or mammalian cell, or prokaryotic cell, such as E.coli. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures, e.g., ligation polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The cytoplasmic domains of the CAR molecules described herein include one or more co-stimulatory domains and one or more signaling domains. The co-stimulatory and signaling domains are used to transmit signals and activate molecules, such as T cells, in response to antigen binding. One or more co-stimulatory domains is/are derived from the stimulatory molecule and/or co-stimulatory molecule, and the signaling domain is derived from a primary signaling domain, e.g. a CD3- ζ domain. In embodiments, the signaling domain further comprises one or more functional signaling domains derived from a co-stimulatory molecule. In embodiments, the costimulatory molecule is a cell surface molecule (other than the antigen receptor or ligand thereof) required to activate a cellular response to an antigen.

In embodiments, the costimulatory domain comprises CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, intracellular domain of CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or any combination thereof. In embodiments, the signaling domain comprises a CD 3-zeta domain derived from a T cell receptor.

In embodiments, the cytoplasmic domain of the CAR comprises only one or more stimulation domains and no signaling domain.

The CAR molecule also includes a transmembrane domain. Incorporation of the transmembrane domain in the CAR molecule stabilizes the molecule. In embodiments, the transmembrane domain of the CAR molecule is the transmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of the CAR, a spacer domain may be incorporated. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide used to attach a transmembrane domain to an extracellular domain or cytoplasmic domain on a polypeptide chain. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids.

Lymphocyte or T cell response in a subject refers to cell-mediated immunity associated with helper cells, killer cells, regulatory cells, and other types of T cells. For example, T cell responses may include activities such as assisting other WBCs in the immune process as well as identifying and destroying virus-infected cells and tumor cells. The T cell response in a subject can be measured by various indicators, such as a number of virally infected cells and/or tumor cells killed by the T cells, the amount of cytokines and/or tumor cells released by the T cells when co-cultured with the virally infected cells, the proliferation level of the T cells in the subject, a phenotypic change of the T cells, such as a change in memory T cells, and the level lifetime or lifespan of the T cells in the subject.

In embodiments, methods of enhancing T cell responses treat a subject in need thereof, e.g., a subject diagnosed with a tumor. The term tumor refers to a bulk tumor, which may be a collection of liquid tumors, such as blood or solid matter. Tumors may be malignant (cancerous) or benign. Examples of blood cancers include chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphocytic leukemia and multiple myeloma.

Solid tumors typically do not contain cysts or areas of fluid. The major types of malignant solid tumors include sarcomas and carcinomas. Sarcomas are tumors that develop in soft tissue cells called mesenchymal cells, which can be found in blood vessels, bones, adipose tissue, ligament lymphatic vessels, nerves, cartilage, muscles, ligaments or tendons, whereas carcinomas are tumors formed in epithelial cells, found in skin and mucous membranes. The most common types of sarcomas include undifferentiated polymorphous sarcomas, which involve soft tissues and bone cells; leiomyosarcoma, which includes smooth muscle cells lining the blood vessels, gastrointestinal tract, and uterus; osteosarcoma involving bone cells and liposarcoma involving adipocytes. Some examples of sarcomas include ewing's sarcoma, rhabdomyosarcoma, chondrosarcoma, mesothelioma, fibrosarcoma, and glioma.

Five of the most common cancers include adrenal cancer, which involves organs that produce fluid or mucus, such as the breast and prostate; basal cell carcinoma, including cells of the outermost layer of the skin, such as skin carcinoma; squamous cell carcinoma, involving basal cells of the skin; and transitional cell carcinoma affecting transitional cells of the urinary tract, including the bladder, kidneys and ureters. Examples of the cancer include thyroid cancer, breast cancer, prostate cancer, lung cancer, intestinal cancer, skin cancer, pancreatic cancer, liver cancer, renal cancer, and bladder cancer, and bile duct cancer.

The methods described herein can be used to treat a subject diagnosed with cancer. The cancer may be a blood cancer or may be a solid tumor, such as a sarcoma or carcinoma. The method of treatment comprises administering to the subject an effective amount of T cells comprising a first antigen binding domain that binds to a cell surface molecule of WBCs and a second antigen binding domain that binds to an antigen that is different from the cell surface molecule of WBCs to provide a T cell response. In embodiments, enhancing a T cell response in a subject comprises selectively enhancing proliferation of T cells expressing a first antigen binding domain and a second antigen binding domain in vivo.

Embodiments described herein relate to in vitro methods for preparing modified cells. The method may comprise obtaining a cell sample from a subject. For example, the sample may comprise T cells or T cell progenitors. The method may further comprise transfecting the cell sample with DNA encoding at least the CAR, and culturing the CAR cell population ex vivo in a medium that selectively enhances proliferation of CAR-expressing T cells.

In embodiments, the sample is a cryopreserved sample. In embodiments, the cell sample is from umbilical cord blood or a peripheral blood sample from the subject. In embodiments, the cell sample is obtained by apheresis or venipuncture. In embodiments, the cell sample is a subpopulation of T cells.

Some embodiments relate to an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding a therapeutic agent that is or comprises a composition of at least one IL-2, IL-6, IL-7, IL-15, IL-17 and IL-23. In some embodiments, the therapeutic agent is or comprises Eome, TRAF6, IL12, IL2, IL18, IL23, AQP9, runx3, AMPK or BCL-2.

Some embodiments relate to an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding a therapeutic agent that is or comprises at least one TNFRSF superfamily member receptor activating antibody or a membrane-bound form thereof, a TNFRSF superfamily member ligand or a membrane-bound form thereof, a different chemokine or a membrane-bound form thereof, a chemokine antibody, or a chemokine or membrane receptor antibody-bound form thereof, may correspond to the sequences in table 7 with the ligand of the D28 family. A TNFRSF superfamily member receptor activating antibody or a membrane-bound form thereof, a TNFRSF superfamily member ligand or a membrane-bound form thereof, a chemokine antibody, or a chemokine receptor antibody or a membrane-bound form thereof, or a ligand of the D28 family. For example, the TNFRSF superfamily member receptor may include tumor necrosis factor receptor 1, tumor necrosis factor receptor 2, lymphotoxin beta receptor, CD40, fas receptor, decoy receptor 3, CD27, CD30,4-1BB, death receptor 4, death receptor 5, decoy receptor 1, decoy receptor 2, RANK, osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry medium, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-induced TNFR-related, TROY, death receptor 6, death receptor 3,Ectodysplasin A2 receptor, and the like

In some embodiments, the therapeutic agent is or includes an antibody agent (e.g., a single chain antibody (e.g., scFv), a single domain antibody (e.g., camelid antibody), or a bispecific antibody agent (e.g., bispecific T cells)), in other embodiments, the therapeutic agent is or includes a cytokine.

Some embodiments relate to a population of CAR cells comprising a nucleic acid sequence and an additional nucleic acid sequence, wherein the CAR cells comprise lymphocytes, leukocytes or PBMCs. In some embodiments, the CAR and therapeutic agent are produced in the form of a polyprotein that is cleaved to produce the individual CAR and therapeutic agent molecules. In some embodiments, the polyprotein comprises a cleavable moiety between the CAR and the therapeutic agent, the cleavable moiety comprises a 2A peptide, the 2A peptide comprises P2A or T2A, and/or the CAR and the therapeutic agent are each constitutively expressed. In some embodiments, the CAR cell comprises: a third nucleic acid sequence encoding a further CAR that binds to an antigen other than a CAR, or a further CAR that binds to a solid tumor antigen, and the CAR binds to an antigen of a leukocyte. In embodiments, the therapeutic agent or variant thereof may be recombinantly or synthetically produced. For the synthetic production of therapeutic agents, an automated synthesizer may be used. For recombinant production of the therapeutic agent, a suitable plasmid containing a polynucleotide encoding the therapeutic agent may be introduced into a suitable host cell, a eukaryotic cell, such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell, such as E.coli. Polynucleotides encoding therapeutic agents of interest may be prepared by conventional procedures, such as ligating polynucleotides. The resulting therapeutic agent may be isolated using standard protein purification techniques known in the art.

Some embodiments relate to a pharmaceutical composition comprising a population of CAR cells. Some embodiments relate to methods of eliciting a T cell response and/or treating a tumor in a subject in need thereof, the methods comprising administering to the subject an effective amount of a composition.

Some embodiments relate to a modified cell comprising one or more CARs, wherein the cell is engineered to express and secrete a therapeutic agent that is or comprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23. In some embodiments, the cells are engineered to express a therapeutic agent that binds to the membrane of the modified cells.

Some embodiments relate to a method of eliciting or enhancing a T cell response, treating cancer, or enhancing a cancer treatment, the method comprising: administering an effective amount of a T cell composition comprising one or more CARs, wherein the cells are engineered to express and secrete a therapeutic agent that is or comprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23, and with T cells that do not express or secrete a therapeutic agent.

Some embodiments relate to a method of eliciting or enhancing a T cell response, treating cancer, or enhancing a cancer treatment, the method comprising: administering an effective amount of a composition comprising a T cell population of a CAR; administering an effective amount of a therapeutic agent that is or comprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23, wherein the T cell response is enhanced as compared to administration of CAR T cells without the therapeutic agent. In some embodiments, administering an effective amount of a therapeutic agent comprises intravenous delivery of an amount of human IL-6 in the range of about 0.5-50 μg/kg body weight. In some embodiments, the therapeutic agent is IL-6 or IL-7.

In some embodiments, the method may further comprise monitoring the concentration of the therapeutic agent in the tissue or blood of the subject; if the concentration and/or other parameters of the subject are not under the desired conditions, a therapeutic agent or receptor antagonist of a therapeutic agent (e.g., an antibody) is administered. For example, the parameters may include body temperature levels, CRS levels, and neurotoxicity levels, among others.

In some embodiments, expression and/or secretion of the therapeutic agent may be modulated by an inducible expression system. In some embodiments, the inducible expression system is an rtTA-TRE system that increases or activates expression of the therapeutic agent or combination thereof. In some embodiments, the inducible expression system is the rtTA-TRE system. For example, tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). In some embodiments, the cells in which expression and/or secretion of the therapeutic agent can be modulated and/or modified by the inducible expression system comprise a nucleic acid sequence encoding an inducible suicide system. For example, the inducible suicide system is an HSV-TK system or an inducible caspase-9 system.

In some embodiments, the T cell comprises an additional CAR that binds to a solid tumor antigen, and the CAR binds to an antigen of a leukocyte. In some embodiments, the solid tumor antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL 13-ra 2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-a, erbB2, epCAM, EGFRvIII, PSCA, or EGFR, and the B cell antigen is CD19, CD20, CD22, or BCMA.

In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain binding antigen.

In some embodiments, the intracellular domain comprises a costimulatory signaling region comprising the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28,4-1bb, ox40, CD30, CD40, pd-1, icos, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

In some embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2 (HER 2), mesothelin (MSLN), prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), disialoganglioside 2 (GD 2), interleukin-13 Ra2 (IL 13Ra 2), glypican-3 (GPC 3), carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125 (CA 125), cluster of differentiation 133 (CD 133), fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 1B), mucin 1 (MUC 1), folate receptor- α (FR- α), CD19, FZD10, hr, PRLR, MUC 17, gucy2c, CD207, CD3, CD5, B Cell Maturation Antigen (BCMA) or CD4.

In some embodiments, the modified cell or T cell comprises a dominant negative PD-1 mutant such that the PD-1/PD1-1 signaling pathway of the cell is disrupted.

In some embodiments, the therapeutic agent is present in the modified cell in a recombinant DNA construct, mRNA, or viral vector. In some embodiments, the modified cell comprises a therapeutic agent mRNA encoding the therapeutic agent, and the mRNA is not integrated into the genome of the modified cell. In some embodiments, the therapeutic agent mRNA may be introduced (e.g., electroporated) into the modified cell such that expression and/or secretion of the therapeutic agent is transient. Synthetic mRNA can be injected to achieve transient gene expression. For example, the therapeutic agent provided by the mRNA is transient such that release of the therapeutic agent is controllable, particularly for pro-inflammatory cytokines, such as: IFN-gamma, IL-4, TNF alpha, IL-8, IL-5, IL-6, GM-CSF and MIP-1 alpha.

In some embodiments, the modified cell comprises a nucleic acid sequence comprising or an isolated nucleic acid sequence comprising a promoter comprising a binding site for a transcriptional regulator (e.g., a transcription factor) that regulates expression of the therapeutic agent in the cell. Examples of nucleic acid sequences or isolated nucleic acid sequences are provided in table 7, these constructs can be placed in a vector (e.g., a lentiviral vector) in either the forward or reverse direction. In some embodiments, the transcriptional modulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB. In some embodiments, the promoter is responsive to a transcriptional regulator. In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a therapeutic agent such that the promoter drives expression of the therapeutic agent in the cell. In some embodiments, the therapeutic agent is linked to a specific promoter to induce expression of the therapeutic agent under desired conditions. Promoters are divided into two parts, a specific regulatory region containing the binding site for the transcription factor, plus a minimal promoter. In some embodiments, more information about NFAT corresponding to the sequences listed in table 7 for promoters and binding sites can be found in WO2018006882, which is incorporated herein by reference.

Some embodiments relate to an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding a therapeutic agent. For example, the therapeutic agent is or comprises IL-6 or IFN-gamma, or a combination thereof. For example, the therapeutic agent is IL-15 or IL-12, or a combination thereof. Some embodiments relate to a population of CAR cells comprising a nucleic acid sequence and an additional nucleic acid sequence, wherein the CAR cells comprise lymphocytes, leukocytes or PBMCs. In embodiments, the CAR cell population of some embodiments, wherein the CAR and therapeutic agent are produced in the form of a polyprotein that is cleaved to produce the individual CAR and therapeutic agent molecules. In embodiments, the polyprotein comprises a cleavable moiety between the CAR and the therapeutic agent, the cleavable moiety comprises a 2A peptide, the 2A peptide comprises P2A or T2A, and/or the CAR and the therapeutic agent are each constitutively expressed. In embodiments, the CAR cell comprises: a third nucleic acid sequence encoding a further CAR that binds to an antigen other than a CAR, or a further CAR that binds to a solid tumor antigen, and the CAR binds to an antigen of a leukocyte. Some embodiments relate to a pharmaceutical composition comprising a population of CAR cells. Some embodiments relate to methods of eliciting a T cell response and/or treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a composition of some embodiments. In embodiments, the CAR cell, modified cell, cell is a T cell, NK cell, macrophage or dendritic cell. For example, the CAR cell, modified cell, cell is a T cell.

In embodiments, the additional nucleic acid sequence comprises a first nucleic acid sequence encoding IL6 and a second nucleic acid sequence encoding IFN- γ, and the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES element or a third nucleic acid. A sequence encoding a 2A peptide. In embodiments, the additional nucleic acid sequence is or comprises SEQ ID NO:21 or 22, or a combination thereof. In embodiments, expression of the additional nucleic acid sequence is modulated by a conditional expression system such that the therapeutic agent is expressed in response to binding of the target antigen. In embodiments, expression of the additional nucleic acid sequence is regulated by a SynNotch polypeptide.

Some embodiments relate to FC fusion proteins associated with small proteins (e.g., cytokines) as described above. In embodiments, the therapeutic agent may comprise an FC fusion protein. For example, a cytokine such as IL-15, IFN-gamma or IL-6 may be linked to one or more immunoglobulin Fc domains. In embodiments, the Fc domains fold independently and may improve the solubility and stability of small proteins in vitro and in vivo. In embodiments, the Fc region allows for easy cost-effective purification by protein-G/a affinity chromatography during manufacturing. In embodiments, FC fusion proteins may be modified to polymerize into well-defined complexes containing multiple small proteins. In embodiments, the fusion protein can be expressed and secreted by a modified cell (e.g., CAR T cell) for use in treating a subject having cancer and/or other diseases. In embodiments, administration of the fusion protein can be combined with treatment of CAR T cells expressing and secreting the fusion protein. For example, a method for enhancing a T cell response and/or treating a subject having cancer or other disease can include administering to the subject a fusion protein associated with a small protein (e.g., IFN- γ) and administering an effective amount of a composition comprising a CAR and expressing and secreting the fusion protein associated with the small protein into a T cell population in the subject. In embodiments, administration of the fusion protein can enhance expansion of the CAR T cells at an early stage of CAR T treatment (e.g., 1,2,3,4,5, or 6 days after infusion of the CAR T cells). For example, the fusion protein can be administered to the subject 1,2,3,4,5, or 6 days after the CAR T cells are infused. In embodiments, the method can include administering to the subject a fusion protein associated with a small protein (e.g., IFN- γ), and administering an effective amount of a composition comprising a T cell population of the CAR without expressing or secreting the fusion protein. Correlating to small proteins in the subject. For example, the fusion protein may be administered to a subject over a predetermined period of time. More information about FC fusion proteins can be found in J Immunol 2004, 172:2925-2934 and EMBO Mol Med2012,4 (10): 1015-1028. Which is incorporated by reference. More information about the administration of therapeutic agents (e.g., cytokines) can be found in J Interferon Cytokine Res 2019,39 (1): 6-21, which is incorporated by reference.

Some embodiments relate to a modified cell comprising one or more CARs, wherein the cell is engineered to express and secrete a therapeutic agent. For example, the therapeutic agent is or comprises IL-6 or IFN-gamma, or a combination thereof. Some embodiments relate to a method of eliciting or enhancing a T cell response, treating cancer, or enhancing a cancer treatment, the method comprising: administering an effective amount of a T cell composition comprising one or more CARs, wherein the cells are engineered to express and secrete a therapeutic agent. For example, the therapeutic agent is or comprises IL-6 or IFN-gamma, or a combination thereof. In embodiments, the therapeutic agent is a small protein associated with IL-6 or IFN-gamma. For example, administration of IL-15 to a subject can increase the concentration of IL-6 and IFN-gamma in the blood of the patient by up to 50-fold. Some embodiments relate to a method of eliciting or enhancing a T cell response, treating cancer, or enhancing a cancer treatment, the method comprising: administering an effective amount of a composition comprising a T cell population of a CAR; and administering an effective amount of the therapeutic agent. For example, the therapeutic agent is or comprises IL-6 or IFN-gamma, or a combination thereof. In embodiments, the CAR cell, modified cell, cell is a T cell, NK cell, macrophage or dendritic cell. For example, the CAR cell, modified cell, cell is a T cell. Some embodiments relate to methods of enhancing a T cell response and/or treating a subject having cancer or other disease, which may include administering a therapeutic agent (e.g., recombinant or natural IFN- γ) to the subject and administering an effective amount of one of a composition comprising a CAR and expressing and secreting the therapeutic agent to a T cell population of the subject. In embodiments, administration of the therapeutic agent can enhance expansion of the CAR T cells at an early stage of CAR T treatment (e.g., 1,2,3,4,5, or 6 days after infusion of the CAR T cells). For example, the therapeutic agent can be administered to the subject 1,2,3,4,5, or 6 days after the infusion of CAR T cells. In embodiments, the method can include administering a therapeutic agent to a subject and administering an effective amount of a composition comprising a T cell population of a CAR without expressing or secreting the therapeutic agent to the subject. For example, the therapeutic agent may be administered to the subject for a predetermined period of time. In embodiments, the therapeutic agent may be modified such that the biological and/or pharmacological properties of the therapeutic agent may be enhanced. For example, hybrid FC fusion techniques may be implemented as solubility and/or stability of the active ingredient of the therapeutic agent.

In embodiments, the therapeutic agent may be an isolated native or recombinant human cytokine. For example, recombinant human IL-15 may be administered for a predetermined period of 3 mcg/kg/day and 1 mcg/kg/day by daily bolus infusion. Recombinant human IFN-gamma can be administered at a dose of 2 million units per day for 5 days per week over a predetermined period of time. In embodiments, administering an effective amount of the therapeutic agent comprises administering an effective amount of the therapeutic agent such that the concentration of IL-6 and/or IFN- γ in the blood of the subject can be increased 5-1000 fold (e.g., 50 times). For example, the therapeutic agent comprises IL-15.

In embodiments, the T cell response is enhanced as compared to administration of a T cell that does not express or secrete the therapeutic agent, or as compared to administration of a CAR T cell that does not administer the therapeutic agent.

In embodiments, the cells whose expression and/or secretion is modulated and/or modified by the inducible expression system comprise a nucleic acid sequence encoding an inducible suicide system. In embodiments, the inducible expression system is the rtTA-TRE system. In embodiments, the inducible suicide system is an HSV-TK system or an inducible caspase-9 system.

In embodiments, the concentration of IL-6 in the blood of a subject ranges from 60 to 5000pg/mL,200-5000pg/mL, or 2000-5000pg/mL. In embodiments, the concentration of IFN- γ in the blood of a subject ranges from 20 to 5000pg/mL, from 200 to 5000pg/mL, or from 500 to 5000pg/mL. In embodiments, administering an effective amount of a therapeutic agent includes intravenous delivery of an amount of human IL-6 in the range of about 0.5-50 μg/kg body weight. In embodiments, the modified cells can express a therapeutic agent such that the concentration of IL-6 and/or IFN- γ in the blood of a subject can be increased 5-1000 fold (e.g., 50 fold). For example, the therapeutic agent comprises IL-15. More detailed information about IFN-gamma clinical use can be found in Cancer Med 2018,7:4509-4516, which is incorporated by reference.

In embodiments, the modified cell or T cell comprises an additional CAR that binds to a solid tumor antigen, and the CAR binds to an antigen of a leukocyte. In embodiments, the solid tumor antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA, or EGFR and B cell antigen is CD19, CD20, CD22, or BCMA.

In embodiments, the modified cell or T cell comprises a dominant negative PD-1. In embodiments, the modified cell or T cell comprises a modified PD-1 that lacks a functional PD-1 intracellular domain.

In embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain binding antigen. In embodiments, the intracellular domain comprises a costimulatory signaling region comprising an intracellular domain selected from the group consisting of CD27, CD28,4-1bb, ox40, CD30, CD40, pd-1, icos, lymphocyte function-associated costimulatory molecules. Antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and combinations thereof. In embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2 (HER 2), mesothelin (MSLN), prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), disialoganglioside 2 (GD 2), interleukin-13 Ra2 (IL 13Ra 2), glypican-3 (GPC 3), carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125 (CA 125), cluster of differentiation 133 (CD 133), fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 1B), mucin 1 (MUC 1), folate receptor- α (FR- α), CD19, FZD10, TSHR, PRLR, MUC17, gucy2c, CD207, CD3, CD5, B Cell Maturation Antigen (BCMA) or CD4.

In embodiments, the therapeutic agent is present in the modified cell in a recombinant DNA construct, mRNA, or viral vector. In embodiments, the modified cell comprises a therapeutic agent mRNA encoding the therapeutic agent, and the mRNA is not integrated into the genome of the modified cell. In embodiments, the modified cell comprises a nucleic acid sequence comprising or comprising an isolated nucleic acid sequence, and the promoter comprises a binding site for a transcriptional regulator that regulates expression and/or secretion of the therapeutic agent in the cell. In embodiments, the transcriptional modulator is or includes Hif1a, NFAT, FOXP3 and/or NFkB. In embodiments, the promoter is responsive to a transcriptional regulator. In embodiments, the promoter is operably linked to a nucleic acid sequence encoding a therapeutic agent such that the promoter drives expression and/or secretion of the therapeutic agent in the cell. In embodiments, the promoter comprises SEQ ID Nos: 23-26.

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

Embodiments described herein also relate to a modified T cell comprising a CAR, wherein the T cell comprises a nucleic acid sequence comprising a nucleic acid sequence encoding IL-6 and a nucleic acid sequence encoding IFN- γ in tandem, the T cell engineered to express and secrete IL-6 and INF- γ when the T cell is activated, wherein the CAR comprises an extracellular domain, a transmembrane domain, an intracellular domain, and an extracellular domain binding antigen.

In embodiments, the modified T cells have no attenuation in tumor-inhibiting function compared to T cells that do not express and/or secrete IL-6 and INF-gamma. Here, certain cytokines (e.g., IL-6 and INF-gamma) are selected for expression or overexpression in T cells. These cytokines do not at least impair the killing function and/or ability to inhibit tumor cells. Because not all cytokines can be expressed and secreted by T cells without reducing their function of killing tumor cells and/or inhibiting tumor growth. Reports indicate tumor promotion by certain cytokines. For example, IL-10 produced by TAM can attenuate an anti-tumor response by inhibiting the function of APC and subsequently blocking T cell effector functions such as cytotoxicity (Manning, MH, zhu, Z., xiao, H., bai, Q., wakefield, MR, fang, Y., cancer Lett.2015Oct 28;367 (2): 103-7). Studies in mouse tumor models have shown that IL-10 can inhibit tumor-infiltrating DC maturation and its production of IL-12 to stimulate Th1 cells unless IL-10 signaling is simultaneously blocked (Vicari AP, chiodoni C, vaure C, ait-Yahia S, dercamp C, matsos F, reynard O, taverne C, merle P, colombo MP, O' Garra A, trincheri G, caux C.J Exp Med.2002;196:541-549. Reversing tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotides and anti-interleukin 10 receptor antibodies (J.Exp. Med.196, 541-549.) for example, studies have shown that TGF-beta may be an effective inhibitor of T cell proliferation (Kehrl JH, wakefield LM, roberts AB, jakowlew S, alvarez-Mon M, derynck R, sporn MB, fauci AS. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med.1986May 1;163 (5): 1037-1050). Several mechanisms drive TGF-beta-mediated inhibition of T cell proliferation, including inhibition of IL-2 production, down-regulation of C-myc and up-regulation of cyclin-dependent kinase inhibitors (Li MO, wan YY, sanjabi S, robertson AK, av, flbertson 3-beta regulation of immune responses, annu Renu 2006. 146; 24-99. ANG. 84. 4. More important factors in the growth of the TGF-beta. 4. Marv, and expansion of human TGF-beta. 4. More important factors, TGF-beta. More specifically, TGF-beta. Mediated inhibition of T cell proliferation, can guide normal leukocyte migration. They are also involved in leukocyte development and pathogenesis of many diseases. In addition, some chemokine concentration gradients play an important role in T cell migration within the junction. Overexpression of these chemokines disrupts T cell migration, thereby impairing CAR T cell therapy of solid tumors. Without proper migration, T cells may not reach tumor cells. For example, over-expression of chemokine CCL21 has been reported to disrupt T cell migration (Christopherson KW and, campbell JJ, hromas RA. Transgenic overexpression of the CC chemokine CCL displays T-cell migration. Blood.2001Dec 15;98 (13): 3562-8). Thus, in embodiments, cytokines that are over-expressed or expressed in the modified cells do not include CCL21, IL-10, and/or TGF- β. In embodiments, cytokines that are overexpressed or expressed in the modified cells do not include IL-10 and/or TGF- β. In embodiments, cytokines that are over-expressed or expressed in the modified cells do not include TGF- β.

In embodiments, the modified T cells have no attenuation in tumor-inhibiting function compared to T cells that do not express and/or secrete IL-6 and INF-gamma.

In embodiments, the modified T cells do not express and/or secrete IL-10 and/or TGF- β.

In embodiments, the CAR binds to a solid tumor antigen. Certain cytokines may be overexpressed or expressed in T cells to enhance CAR T therapy for treating tumors. However, some cytokines (e.g., IL-6) cannot be over-expressed or expressed in T cells to treat blood tumors. IL-6 is a major factor in causing CAR T to treat severe CRS of hematological tumors such as ALL and NHL. Thus, IL-6 can be overexpressed or expressed in T cells to treat solid tumors, as few reports have shown that CART treats severe CRS of solid tumors.

In embodiments, the modified T cell comprises a nucleic acid encoding SEQ ID NO:21 and 22.

In embodiments, the CAR binds tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA, or EGFR. In embodiments, the intracellular domain comprises a costimulatory signaling region comprising the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and combinations thereof. In embodiments, the antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1. GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA, or EGFR.

In embodiments, the nucleic acid sequence comprises a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of the transcriptional regulator. In embodiments, the transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, or NFkB. In embodiments, the promoter is responsive to a transcriptional regulator. In embodiments, the promoter may be linked to the nucleic acid sequence such that the promoter drives expression and/or secretion of IL-6 and INF-gamma in the T cells. In embodiments, the promoter comprises SEQ ID Nos: 23-26.

In embodiments, the polypeptide comprises a sequence encoding SEQ ID NO:21 and 22, and SEQ ID NO:27 such that IL-6 and INF-gamma are expressed and secreted when T cells are activated.

In embodiments, the CAR, IL-6 and INF- γ are produced in the form of a multimeric protein that is cleaved to produce the CAR, IL-6 and INF- γ alone, and there is a cleavable moiety between the CAR, IL-6 and INF- γ, the cleavable moiety comprising a 2A peptide, the 2A peptide comprising P2A or T2A.

Further, embodiments described herein relate to the use of a composition to elicit a T cell response in a subject in need thereof and/or to treat a tumor in a subject, the method comprising administering to the subject an effective amount of the composition, wherein the composition comprises the modified cells described above. The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present disclosure should in no way be construed as limited to the following exemplary embodiments and examples, but rather should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

Drawings

The specific embodiments are described with reference to the accompanying drawings. The use of the same reference numbers in different figures indicates similar or identical items.

FIG. 1 shows the results of flow cytometry assays for T cells expressing various proteins.

FIG. 2 shows the copy number of T cells expressing various proteins.

FIG. 3 shows IL-6 release in response to CD3/CD28 Dynabeads activation.

FIG. 4 shows IL-6 release in response to co-culture with nalm6 cells.

FIG. 5 shows IFN-gamma (i.e., IFNg) release in response to CD3/CD28 Dynabeads activation.

FIG. 6 shows IFN-gamma release in response to co-culture with nalm6 cells.

Figure 7 shows toxicity assays for CAR T cells.

Figures 8 and 9 show other IFN- γ release in response to co-culture with nalm6 cells.

FIGS. 10 and 11 show IL-12 and IFN-gamma release in response to CD3/CD28 Dynabeads activation.

FIGS. 12 and 13 show IL-6 and IFN-gamma release in response to CD3/CD28 Dynabeads activation.

Fig. 14 shows a schematic example of the structure of a CAR and a therapeutic agent.

Detailed Description

The invention is further described below in connection with specific embodiments.

The following are exemplary embodiments:

1. an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) and an additional nucleic acid sequence encoding a therapeutic agent comprising IL-6 or IFN- γ, or a combination thereof.

2. A population of CAR cells comprising the nucleic acid sequence of embodiment 1 and an additional nucleic acid sequence, wherein the CAR cells comprise lymphocytes, leukocytes or PBMCs.

3. The CAR cell population of embodiment 2, wherein the CAR and therapeutic agent are produced in the form of a polyprotein that is cleaved to produce the individual CAR and therapeutic agent molecules.

4. The CAR cell population of one of embodiments 2-3, wherein the polyprotein comprises a cleavable moiety between the CAR and the therapeutic agent, the cleavable moiety comprises a 2A peptide, the 2A peptide comprises P2A or T2A, and/or the CAR and the therapeutic agent are each constitutively expressed.

5. The CAR cell population of one of embodiments 2-4, wherein the CAR cells comprise: a third nucleic acid sequence encoding an additional CAR that binds to an antigen that is different from the antigen to which the CAR binds, or an additional CAR binds to a solid tumor antigen, the CAR binding to an antigen of a leukocyte.

6. A pharmaceutical composition comprising the CAR cell population of one of embodiments 2-5.

7. A method of eliciting a T cell response and/or treating a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of embodiment 6.

8. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, or a method of one of embodiments 1-7, wherein the additional nucleic acid sequence comprises a first nucleic acid sequence encoding IL-6 and a second nucleic acid sequence encoding a second nucleic acid sequence. IFN-gamma and the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES element or a third nucleic acid sequence encoding a 2A peptide.

9. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, or a method of one of embodiments 1-8, wherein the additional nucleic acid sequence is or comprises SEQ ID NO:21 or 22, or a combination thereof.

10. An isolated nucleic acid sequence, CAR cell population, pharmaceutical composition, or method of one of embodiments 1-9, wherein expression of the additional nucleic acid sequence is modulated by a conditional expression system such that the therapeutic agent is expressed in response to binding of the target antigen, or expression of the additional nucleic acid sequence is modulated by a SynNotch polypeptide.

11. A modified cell engineered to express and secrete a therapeutic agent that is or comprises IL-6 or IFN- γ, or a combination thereof.

12. The modified cell of embodiment 11, wherein the modified cell comprises one or more CARs.

13. A method of eliciting or enhancing a T cell response, treating cancer or enhancing cancer treatment, the method comprising: administering an effective amount of a T cell composition comprising one or more CARs, wherein the cells are engineered to express and secrete a therapeutic agent that is or comprises IL-6 or IFN- γ, or a combination thereof.

14. A method of eliciting or enhancing a T cell response, treating cancer or enhancing cancer treatment, the method comprising: administering an effective amount of a composition comprising a T cell population of a CAR; an effective amount of a therapeutic agent of IL-6 or IFN-gamma or a combination thereof, is administered.

15. The modified cell or method of one of embodiments 7,13 and 14, wherein the T cell response is enhanced as compared to administration of a T cell that does not express or secrete the therapeutic agent, or the T cell response is enhanced as compared to administration of a CAR T cell that does not administer the therapeutic agent.

16. The modified cell or method of any of embodiments 11-15, wherein expression and/or secretion of the therapeutic agent is modulated by the inducible expression system and/or the modified cell comprises a nucleic acid sequence encoding an inducible suicide system.

17. The modified cell or method of any of embodiments 11-15, wherein the concentration of IL-6 in the blood of IL-6 ranges from 60 to 5000pg/mL, from 200 to 5000pg/mL, or from 2000 to 5000pg/mL.

18. The modified cell or method of any of embodiments 11-15, wherein the concentration of IFN- γ in the blood ranges from 20 to 5000pg/mL, from 200 to 5000pg/mL, or from 500 to 5000pg/mL.

19. The modified cell or method of any of embodiments 11-18, wherein administering an effective amount of the therapeutic agent comprises intravenous delivery of human IL-6 in an amount of about 0.5-50ug/kg body weight.

20. The modified cell or method of one of embodiments 11-19, wherein the modified cell or T cell comprises an additional CAR that binds to a solid tumor antigen, and the CAR binds to an antigen of a leukocyte.

21. The modified cell or method of embodiment 20, wherein the solid tumor antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, psma, ror1, vegfr-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA or EGFR, and the B cell antigen is CD19, CD20, CD22 or BCMA.

22. The modified cell or method of any one of embodiments 11-21, wherein the modified cell or T cell comprises dominant negative PD-1.

22. The modified cell or method of any of embodiments 11-21, wherein the modified cell or T cell comprises a modified PD-1 that lacks a functional PD-1 intracellular domain.

23. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or the method of one of embodiments 1-23, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain binding antigen.

24. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or a method of one of embodiments 1-23, wherein the intracellular domain comprises a costimulatory signaling region comprising the intracellular domain of a costimulatory molecule. Selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and combinations thereof.

25. An isolated nucleic acid sequence, CAR cell population, pharmaceutical composition, modified cell, or method of one of embodiments 1-24, wherein the antigen is Epidermal Growth Factor Receptor (EGFR), epidermal growth variant III, factor receptor (EGFRvIII), human epidermal growth factor receptor 2 (HER 2), mesothelin (MSLN), prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), disialoganglioside 2 (GD 2), interleukin-13 Ra2 (IL 13Ra 2), phospholipidin-3 (GPC 3), carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125 (CA 125), cluster of differentiation 133 (CD 133), fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 1B), mucin 1 (MUC 1), folate receptor- α (FR- α), CD19, zd10, TSHR, PRLR, MUC17, GUCY2C, CD207, CD3, CD5, B Cell Maturation Antigen (BCMA) or CD4.

26. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or a method of one of embodiments 1-25, wherein the therapeutic agent is present in the modified cell, mRNA, or viral vector as a recombinant DNA construct.

27. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or the method of one of embodiments 1-26, wherein the modified cell comprises a therapeutic mRNA encoding a therapeutic agent, and the mRNA is not integrated into the genome of the modified cell.

28. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or a method of one of embodiments 1-27, wherein the modified cell comprises a nucleic acid sequence comprising or an isolated nucleic acid sequence comprising a promoter comprising a transcriptional regulator binding site that regulates expression and/or secretion of a therapeutic agent in the cell.

29. An isolated nucleic acid sequence, CAR cell population, pharmaceutical composition, modified cell, or method of embodiment 28, wherein the transcriptional regulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

30. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or the method of embodiment 28, wherein the promoter is responsive to a transcriptional regulator.

31. An isolated nucleic acid sequence, CAR cell population, pharmaceutical composition, modified cell, or method of embodiment 28, wherein the promoter is operably linked to a nucleic acid sequence encoding a therapeutic agent such that the promoter drives expression and/or secretion of the therapeutic agent in the cell.

32. An isolated nucleic acid sequence, a population of CAR cells, a pharmaceutical composition, a modified cell, or a method of one of embodiments 28-31, wherein the promoter comprises the nucleic acid sequence of SEQ ID NOs: 23-26.

33. A modified cell comprising nucleic acid sequences encoding IL-6 and IFN- γ, expression of IL-6 and IFN- γ being regulated by the transcriptional regulator NFAT.

34. The modified cell of embodiment 33, wherein the modified cell is an NK, T or macrophage.

35. The modified cell of embodiment 34, wherein the modified cell comprises a CAR, a genetically modified TCR, or a selected TCR that targets a tumor cell.

34. The modified cell of embodiment 33, wherein the nucleic acid sequence comprises SEQ ID NO:28 and the sequence encoding SEQ ID NO:21 and 22.

35. A modified cell comprising a CAR, wherein the cell is engineered to express and secrete a therapeutic agent, the cell comprising a nucleic acid sequence encoding such a therapeutic agent, the nucleic acid sequence comprising. Such therapeutic agents are or comprise IL-6 or IFN- γ or a combination thereof, wherein the CAR comprises an extracellular domain, a transmembrane domain, an intracellular domain, an extracellular domain binding antigen.

36. The modified cell of embodiment 35, wherein the therapeutic agent comprises IL-6 and IFN- γ, or the modified cell comprises a nucleic acid encoding SEQ ID NO:21 and 22.

37. The modified cell of one of examples 35 and 36, wherein the CAR binds tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA or EGFR, B cell antigen is CD19, CD20, CD22 or BCMA.

38. The modified cell of one of embodiments 35-37, wherein the intracellular domain comprises a costimulatory signaling region comprising the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and combinations thereof.

39. The modified cell of one of examples 35-38, wherein the antigen is Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2 (HER 2), mesothelin (MSLN), prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), disialoganglioside 2 (GD 2), interleukin-13 Ra2 (IL 13Rα2), glypican-3 (GPC 3), carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125 (CA 125), cluster of differentiation 133 (CD 133), fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 1B), mucin 1 (MUC 1), folate receptor- α (FR- α), CD19, FZD10, TSHR, PRLR, muc17, GUCY2C, CD207, CD3, CD5, B Cell Maturation Antigen (BCMA) or CD4.

40. The modified cell of any one of embodiments 35-39, wherein the therapeutic agent is present in the modified cell, in a recombinant DNA construct, in an mRNA, or in a viral vector.

41. The modified cell of any one of embodiments 35-40, wherein the modified cell comprises a nucleic acid sequence, the nucleic acid sequence or the isolated nucleic acid sequence comprises a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of the transcriptional regulator.

42. The modified cell of example 41, wherein the transcriptional regulator is or comprises Hif1a, NFAT, FOXP3 or NFKB.

43 the modified cell of example 41, wherein the promoter is responsive to a transcriptional regulator.

44. The modified cell of embodiment 41, wherein the promoter is operably linked to a nucleic acid sequence encoding the therapeutic agent such that the promoter drives expression and/or secretion of the therapeutic agent in the cell.

45. The modified cell of example 7, wherein the promoter comprises SEQ ID NOs: 23-26.

46. The modified cell of one of embodiments 35-45, wherein the modified cell is a modified T cell comprising a nucleic acid encoding SEQ ID NO:21 and 22, and SEQ ID NO:27 such that IL-6 and INF-gamma are expressed and secreted when T cells are activated.

47. The modified cell of example 47, wherein the CAR and the therapeutic agent are produced in the form of a multimeric protein that is cleaved to produce separate CAR and therapeutic agent molecules, and there is a cleavable moiety between the CAR and the therapeutic agent, the cleavable moiety comprising a 2A peptide, the 2A peptide comprising P2A or T2A.

48. Use of a composition to elicit a T cell response and/or to treat a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition, wherein the composition comprises the modified cells of examples 35-47.

49. The composition of example 48 for use in a medicament for one or more of the following: stimulating a T cell-mediated immune response to a target cell population or tissue in a human, providing anti-tumor immunity in a human, treating a human with cancer, preparing a medicament for producing a sustained population of genetically engineered T cells in a human diagnosed with cancer, and expanding the population of genetically engineered T cells including a population of progeny T cells in a human diagnosed with cancer.

50. The composition of embodiment 48, wherein said sustained population of modified cells comprises T cells comprising memory T cells, and wherein said sustained population of T cells persists in said human at least three after administration.

51. A composition comprising a modified T cell, the T cell comprising a CAR, wherein the T cell comprises a nucleic acid sequence comprising (1) a nucleic acid sequence encoding IL-6 and/or (2) a nucleic acid sequence encoding IFN- γ, the T cell engineered to express and secrete IL-6 and or INF- γ when the T cell is activated, wherein the CAR comprises an extracellular domain, a transmembrane domain, an intracellular domain, an extracellular domain binding antigen, and the CAR binds to a solid tumor antigen.

52. The composition of embodiment 51, wherein the modified T cell comprises (1) a nucleic acid sequence encoding IL-6 and/or (2) a nucleic acid sequence encoding IFN- γ in tandem.

53. The composition of embodiment 51, wherein the modified T cell comprises a nucleotide sequence encoding SEQ ID NO:21 and 22.

54. The composition of one of embodiments 51-53, wherein the CAR binds tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA, or EGFR.

55. The composition of any one of embodiments 51-54, wherein said intracellular domain comprises a costimulatory signaling region comprising the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and combinations thereof and/or said antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, ephA2, FAP, GPC3, IL13-Rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, erbB2, epCAM, EGFRvIII, PSCA or EGFR.

56. The composition of any one of embodiments 51-55, wherein the nucleic acid sequence comprises a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of the transcriptional regulator.

57. The composition of example 56, wherein the transcriptional regulator is or comprises Hif1a, NFAT, FOXP3 or NFKB.

58. The composition of example 56, wherein the promoter is responsive to a transcriptional regulator.

59. The composition of embodiment 56, wherein said promoter is operably linked to the nucleic acid sequence such that said promoter drives expression and/or secretion of IL6 and infγ in said T cells.

60. The composition of embodiment 56, wherein the promoter comprises SEQ ID NOs: 23-26.

61. A composition of one of examples 51-60 comprising a polypeptide encoding SEQ ID NO:21 and 22, and SEQ ID NO:27 such that IL-6 and INF-gamma are expressed and secreted when T cells are activated.

62. The composition of example 61, wherein the CAR, IL-6 and INF- γ are produced in the form of a multimeric protein that is cleaved to produce the CAR, IL-6 and INF- γ alone, and a cleavable moiety is present between the CAR, IL-6 and INF- γ, the cleavable moiety comprising a 2A peptide, the 2A peptide comprising P2A or T2A.

63. The composition of one of examples 51-62, wherein the tumor-inhibiting function of the modified T cell is not reduced as compared to a T cell that does not express and/or secrete IL-6 and INF- γ, and the modified T cell does not express and/or secrete TGF- β.

64. Use of a composition to elicit a T cell response and/or to treat a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition, wherein the composition comprises the modified cells of examples 51-63.

65. The composition of one of examples 51-64, which is a pharmaceutical composition comprising an anti-tumor effective amount of modified T cells, wherein said anti-tumor effective amount is preferably 10 per kg body weight of a human in need of such cells ⁴ To 10 ⁹ Or 10 ⁵ To 10 ⁶ Individual cells.

66. The composition of embodiment 65, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

67. The composition of embodiment 65, wherein the pharmaceutical composition further comprises a buffer.

68. The composition of embodiment 67, wherein the buffer is neutral buffered saline or sulfate buffered saline.

69. The composition of embodiment 65, wherein the pharmaceutical composition further comprises a carbohydrate.

70. The composition of embodiment 69, wherein the carbohydrate is selected from the group consisting of glucose, mannose, sucrose, dextran, mannitol.

71. The composition of embodiment 85, wherein the pharmaceutical composition further comprises an injectable freezing medium.

72. The composition of embodiment 71, wherein the injectable freezing medium comprises plasmalyte-A, dextrose, naCl, DMSO, dextran, and human serum albumin.

73. A composition of one of examples 51-72 for use in a medicament for one or more of the following: stimulating a T cell-mediated immune response to a target cell population or tissue in a human, providing anti-tumor immunity in a human, treating a human with cancer, preparing a medicament for producing a sustained population of genetically engineered T cells in a human diagnosed with cancer, and expanding the population of genetically engineered T cells including a population of progeny T cells in a human diagnosed with cancer.

Examples of the invention

Cells expressing chimeric receptors establish antitumor effects in patients with relapsed/refractory acute lymphoblastic leukemia

The clinical trial was designed to evaluate the safety and efficacy of improving autologous T cells used to express CD19 specific CAR/4-1BB/CD3- ζ or CD19 specific CAR/4-1BB/CD3- ζ. The injection criteria were as follows: 1) Age less than 60 years old; 2) Recurrent or refractory cd19+ ALL; 3) Recurrent allogeneic HSCT has no evidence of Graft Versus Host Disease (GVHD), without immunosuppressive treatment; and 4) measurable disease and sufficient performance status and organ function. The protocol was approved by the institutional evaluation committee medical institute first affiliated hospital. All patients provided written informed consent.

The CD19 specific single chain fragment variant (scFv) sequence is derived from Clone FMC63 (see Zola H. Et al Immunol Cell Biol 1991; 69:411-22). The 4-1BB costimulatory domain, the CD 3-zeta signaling domain, and the hinge and transmembrane domains are generated. CART19-4-1BB vectors carrying anti-CD 19 scFv (SEQ ID: 6) and human 4-1BB and CD3- ζ signaling domains were cloned into lentiviral scaffolds as described previously (see Hu Y. Journal of therapeutics & Oncology 2016; 9:70). The anti-CD 19 scFv and the PD-1/CART19-4-1BB vectors of the human 4-1BB and CD3- ζ signaling domains were cloned into the lentiviral backbone.

Transfection of 293T cells with CART19-4-1BB vector and viral packaging plasmid resulted in lentiviruses, which were frozen at-80℃and thawed immediately prior to transduction. Lentiviral supernatants were harvested. CD3+ T cells were isolated and activated as described (see Kalos M. Et al, sci Transl Med 2011; 3:95raf3). Cells were then cultured in X-VIVO 15 medium (Lonza) containing 100U/mL interleukin-2 (IL-2) and transduced with lentiviral supernatant for-48 hours at a high multiplicity of infection (MOI) of 5:1 to 10:1 within 24 hours. CAR-transduced T cells (CD 19-CART cells, followed by "CART 19") were obtained and cultured for 11 days. 3 days prior to dosing, fresh medium was replaced. Thereafter, no manipulation of the cells is performed until the transfusion is carried out. Transduction efficiency was assessed by flow cytometry (FACS) on days 5-7 after lentiviral transduction. The following anti-human antibodies were used: anti-hCD 45APC (BD Bioscience), anti-hCD 3 FITC (BD Bioscience), biotin-labeled goat anti-mouse IgG specific for F (ab') 2 fragments (Jackson immune studies, cat#115-065-072) and PE streptavidin (BD Bioscience). Data acquisition was performed using a CytoFLEX flow cytometer (Beckman). FACS analysis of the transduction efficiency and in vitro cytotoxicity assays of CART19s was performed on each patient prior to CART19s infusion. In addition, CART19 cultures were examined twice for possible contamination with fungi, bacteria, mycoplasma, chlamydia and endotoxins.

On day 8, prepared for CART19s, peripheral Blood Mononuclear Cells (PBMCs) were obtained from the patient using leukapheresis. The first day CART19s infusion was set to day 0. Conditioning therapy for lymphocyte depletion is performed on the patient. Conditioning treatment based on fludarabine and cyclophosphamide varies according to tumor burden in Bone Marrow (BM) and Peripheral Blood (PB). CART19s was directly administered to the patient at increasing doses over a period of 3 consecutive days without any pre-operative medication. CART19s was delivered daily to the hospital, washed, counted, checked for viability, then prepared for administration to the patient, and then closely observed for at least 2 hours. CRS is ranked according to a revised ranking system (see Lee DW et al, blood 2014; 124:188-95). Other toxicities during and after treatment were assessed according to the U.S. national institutes of health adverse event version 4.0 common terminology standard (http:// ctep. Cancer. Gov /). Treatment response was assessed by flow cytometry and morphological analysis. Patients were assessed by chimeric gene expression levels, if possible. The response type is defined as Minimal Residual Disease (MRD) negative, complete response, incomplete count recovery, disease stabilization and progressive disease described in the supplemental material.

Serial BM and PB samples following CART19s infusion were collected in K2EDTA BD vacuum tube (BD). Persistence of CART19s from fresh PB and BM patients was determined by FACS. The number of cyclic CART19 per μl was calculated based on the measured absolute cd3+ T lymphocyte count. Meanwhile, CAR DNA copies were evaluated as another method to determine CART19s amplification and persistence. Genomic DNA was extracted from cryopreserved PB and BM using QIAamp DNA Blood Mini Kit (Qiagen). CAR DNA copies were assessed by quantitative real-time PCR.

The levels of cytokines IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10, IL-17, etc. in serum and CSF are measured in multiplex format according to the manufacturer's instructions. The Mann-Whitney U test was used to compare 2 sets of continuous variables and risk factors that may affect changes in CRS development of grade 3 or grade 4. Fisher's exact test was used to evaluate the effect of classification variables between two groups on class 3 CRS. Correlation was calculated using a rank-based Spearman test. Overall Survival (OS) and leukemia-free survival (LFS) probabilities OS and LFS with MRD negative response were determined by Kaplan-Meier method using all patients in the group. All cited P values are double sided, with P values less than 0.05 considered statistically significant.

CD19+ -RFP and RFP are transduced into K562 by lentivirus to produce CD19-RFP-K562 cells and K562-RFP cells, respectively. Cytotoxic activity of CART19s was measured before infusion by co-culturing with target cells, CD19-RFP-K562 cells or K562-RFP cells, at different ratios of effector cells to target cells (E: T). Target cells were seeded 104 cells per well in 96-well microplates (Nunc) in 50 μlrpmi 1640 medium supplemented with 10% fbs (Gibco). The CD3/CD28 beads were removed and the following indicated E: t ratio mixes effector T cells with target cells in the well. The total volume was 200. Mu.L per well. After 24 hours of incubation, the cells were pipetted up and down in 96-well microwell plates with a multichannel pipette to separate the cells into single cell suspensions. Surviving RFP target cells in each well were photographed, the number of surviving RFP target cells was counted and compared to wells without effector cells. Cell mortality was calculated as (control sample)/control x 100%. Supernatants were also collected and quantified using a human IFN-. Gamma.Valukine ELISA kit (R & D system).

From freezing using QIAamp DNA Blood Mini Kit (Qiagen)Genomic DNA was extracted from the stored peripheral blood and bone marrow. Quantitative real-time PCR was performed in triplicate using ABI 2 x TaqMan Universal Master Mix with amprase UNG (Applied Biosystems) in 7500 real-time PCR systems (Applied Biosystems). Copy number per microgram of genomic DNA was calculated from a standard curve containing 10-fold serial dilutions of purified CAR plasmid at 102-108 copies/. Mu.l. Amplification of internal control genes was used for normalization of DNA amounts, as described previously (see

Blood 2012, et al; 120:2032-41 and O' Brien S. Et al, J Clin Oncol 2013;31: 676-83).

Treatment response was assessed by flow cytometry and morphology. Chimeric gene expression levels were assessed in patients, if possible. The type of response is defined as MRD negative, complete response including incomplete count recovery, disease stabilization, and progressive disease, as previously described. MRD negative was defined by flow cytometry as less than 0.01% of myeloid progenitor cells. Complete response is defined as the extra-medullary site of disease with less than 5% of myeloblasts, no circulating burst, no menopausal neutrophil count of 1000/μl or more, and platelets 100,000 μl/μl or more. The complete response with incomplete count recovery is defined as one with cytopenia. Stable disease is defined as a disease that does not meet the criteria for complete response, incomplete count recovery or progressive disease. Progressive disease is defined as poor or unchanged M-state, with an absolute increase in peripheral blood cell count of more than 50%. Following CART19 treatment, patients were followed weekly, with bone marrow examination including morphology, MRD status, chimeric gene expression and CART cell count performed every 4 weeks.

Samples were collected in gel tubes and stored at 4 ℃ until later on the same day by centrifugation. All blood and CSF samples were then centrifuged at 5000rpm for 6 minutes. The supernatant was transferred for subsequent analysis. BD Cytometric Bead Array Human Th1/Th2/Th17 cytokine kit (BD Biosciences), FCAP Array v3.0 software (BD Biosciences) and BD FACS CANTO II (BD Biosciences) were used to measure and analyze cytokines such as IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-gamma and TNF-alpha, and the like.

All BM samples from erythrocyte lysis were used for immunophenotyping on the day of bone marrow aspiration. The antigen expression of the blasts was systematically analyzed by flow cytometry (FACSCalibur flow cytometer, BD Biosciences, san Jose, CA) and phycoerythrin-anthocyanin 7 (PE-Cy 7) using a four-color combination of monoclonal antibodies (mAb) with Fluorescein Isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC). Cell-Quest software (Becton Dickinson Biosciences) was used for data analysis. Monoclonal antibodies were purchased from the following manufacturers: BD Biosciences, CD10-APC, CD19-FITC, CD22-PE, CD34-PE, CD45-PE-Cy7, cyCD79a-PE, surface immunoglobulin (sIg) M-PE, cytoplasmic immunoglobulin cIg) M-APC; beckman Coulter, CD20-APC, sIg-Lamda-FITC, sIg-Kappa-APC.

For the MRD investigation, the combination of mabs was based on the abnormal phenotype of leukemia blast cells diagnosed individually, with at least 50 ten thousand events obtained. MRD results are expressed as a percentage of cells with an abnormal phenotype in nucleated cells. A sensitivity of 0.01% was achieved in all sample analyses. Daily by analysis of Calibrite ^TM Beads and standard blood samples (BD from BD Biosciences ^TM CD-chex for multiplex inspection control or Streck, inc ^TM Plus) to calibrate the instrument settings for quality control.

Patients (3 cases) were treated with CD19-CAR T cells and the results are summarized in Table 1 below. These results indicate that T cells expressing CD19CAR establish anti-tumor effects in r/r ALL patients. In addition, IL-6 and IFN-gamma were significantly elevated in the blood of three patients after T cells that would transfuse CD19CAR, as compared to other factors (tables 1-4). Thus, to help CAR T cells achieve efficacy in the treatment of solid tumors also in hematological tumors, the following cytokines expressed or overexpressed by T cells for the treatment of solid tumors first select IL-6 and ifnγ.

Table 1

Table 2

TABLE 3

Table 4

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Cells express IL-6, IL-12, and or IFN-gamma and its features/functions

FIG. 1 shows flow cytometry assay results of T cells expressing the various proteins shown in FIG. 1. 18. On day 0, peripheral blood from healthy volunteers was taken, cd3+ T cells were sorted, and the following was applied at 1:1 to CD3/CD28 Dynabeads. On day 2, T cells were transfected with lentiviruses including various of the following vectors. Infection with 19CAR according to MOI = infection rate of 10-1; hCD19CAR, hCD19CAR-6xNFAT-GFP, hCD19CAR-6xNFAT-IL6-2a-ifnγ cells were infected according to the infection rate of moi=60-1. On day 3, the medium was changed, lentiviruses removed, and cells were resuspended in fresh medium. Day 7, flow cytometry assays were used to detect CAR expression. 19CAR is a humanized antibody and thus detected with human CAR antibodies. As shown in fig. 1. 19,19CAR-T CAR expression was 23.99%, hCD19CAR-6xNFAT-GFP CAR expression was 25%, and hCD19CAR-6xNFAT-IL6-2a-IFN gamma CAR expression was 17.6%. Flow cytometry assays were performed using human CAR antibodies to detect the expression intensity and expression level of the CAR.

FIG. 2 shows the copy number of T cells expressing various proteins. On day 1, T cells from healthy donors were sorted and activated using CD3/CD28 beads. On day 2, 105 cells were transfected with vector 1230 (MOI 10:1), 6205 (hCD 19 CAR-GFP) (MOI 60:1) and 6221 (hCD 19CAR-6xNFAT-IL6-2a-IFNγ) (MOI 60:1), respectively. On day 3, the cell culture medium was changed. On day 4, the cell number was counted. On days 5,6 and 8, assays for measuring culture factors, CAR copy number, phenotype and expression were performed. On day 8, toxicity assays were performed and culture factors were tested. The copy number is provided in the table below. In this and the following examples, the sequence of 6xNFAT or NFAT is SEQ ID NO 27; the scFv of the TSHR CAR is SEQ ID NO:8, 8; scFv of CD19CAR is SEQ ID 5, aa of il-6 is SEQ ID NO:21,2A is SEQ ID NO:28, aa of ifn- γ is SEQ ID NO:22. the sequences of the other components can be found in table 7.

FIG. 3 shows IL-6 release in response to CD3/CD28 Dynabeads activation. On day 0, peripheral blood from healthy volunteers was taken, cd3+ T cells were sorted, and the following was applied at 1:1 to CD3/CD28 Dynabeads. On day 2, T cells were transfected with lentiviruses including various of the following vectors. Infection with 19CAR according to MOI = infection rate of 10-1; hCD19CAR, hCD19CAR-6xNFAT-GFP, hCD19CAR-6xNFAT-IL6-2a-ifnγ cells were infected according to the infection rate of moi=60-1. On day 3, the medium was changed, lentiviruses removed, and cells were resuspended in fresh medium. On days 5,6 and 8, the release of IL-6 factor was detected using 200. Mu.L of cell supernatant in culture. As shown in FIG. 3, at days 5,6 and 8, 200. Mu.L of the cell supernatant was removed from the medium, releasing IL-6 factor. Every 10 on days 5,6 and 8 ⁴ The amount of IL-6 released by 19CAR and 19CAR-6xNFAT-GFP is 0-10pg/mL.10 ⁴ CD19CAR-6xNFAT-IL6-2A-IFN gamma has IL6 release of 498pg/mL and release on day 5 and 6 is 378pg/mL and release on day 8 is 9.8pg/mL. On days 5 and 6, cells were cultured with CD3/CD28 Dynabeads, activating the cells, activating the NFAT element, allowing IL-6 to be transcribed and IL-6 to be released. However, on day 8, the effect of Dynabeads stimulation had been reduced to lower levels and the cells were not activated. Thus, the NFAT element is turned off and the transcription of IL-6 is disabled. Thus, IL-6 is not released. When CD3/28Dynabeads stimulated T cells, the cells were activated in a short period of time. At this time, the NFAT element causes transcriptional translation of the gene expressed as a result of activation, and the corresponding expressed gene is expressed when the cell is at rest or at a lower activation level, the NFAT element does not initiate transcriptional translation. A gene of interest in an inactive state. Thus, it can be judged whether the NFAT element is active and whether the induction gene is expressed by expressing the target gene in the activated and inactivated state of the CAR-T cell.

FIG. 4 shows IL6 release in response to co-culture with nalm6 cells (see Table 5 below). Cells were cultured to day 8 and then NT cell levels were used to differentiate CAR ratio of CD19 CAR-T cells and CD19CAR-6xNFAT-GFP to CD19CAR-6xNFAT-IL6-2a-ifnγ cells. Will 10 ⁴ Car+ cells and 10 ⁴ Nalm-6 cells were co-cultured or cultured separately. After 24 hours, the supernatant was collected and the amount of released IL-6 was measured. Cells were co-cultured with nalm6 cells and in an activated state. Thus, the NFAT element initiates transcription of IL6 in the activated state, allowing release of IL-6. When CAR-T cells are co-cultured with target cells, the cells are activated because the CAR-T cells recognize membrane proteins on the surface of tumor cells. The NFAT element initiates transcriptional translation of genes expressed as a result of activation. Expressing the corresponding expressed gene. When the cell is at rest or has a low level of activation, the NFAT element does not initiate transcriptional translation of the gene of interest in an inactive state. Thus, it can be judged whether the NFAT element is active and whether the induction gene is expressed by expressing the target gene in the activated and inactivated state of the CAR-T cell.

Table 5

FIG. 5 shows IFN-gamma (i.e., IFNg) release in response to CD3/CD28 Dynabeads activation. On day 0, peripheral blood from healthy volunteers was taken, cd3+ T cells were sorted, and at 1:1 to CD3/CD28 Dynabeads. On day 2, T cells were transfected with lentiviruses including various of the following vectors. Infection with 19CAR according to MOI = infection rate of 10-1; hCD19CAR, hCD19CAR-6xNFAT-GFP, hCD19CAR-6xNFAT-IL6-2a-ifnγ cells were infected according to the infection rate of moi=60-1. Lentiviruses were removed and cells were resuspended in fresh medium. On days 5,6 and 8, IFN-gamma release was detected using 200. Mu.L of cell supernatant from the culture. On days 5 and 6, cells were cultured with CD3/CD28 Dynabeads, cells were activated, NFAT elements were activated, IFN-gamma was transcribed, and IFN-gamma was released. However, on day 8, the effect of Dynabeads stimulation had been reduced to lower levels and the cells were not activated. Thus, the NFAT element is turned off and transcription of IFN- γ is disabled. Thus, IFN-gamma is not released. When CD3/28Dynabeads stimulated T cells, the cells were activated in a short period of time. At this time, the NFAT element causes transcriptional translation of the gene expressed as a result of activation, and the corresponding expressed gene is expressed when the cell is at rest or at a lower activation level, the NFAT element does not initiate transcriptional translation. A gene of interest in an inactive state. Thus, it can be judged whether the NFAT element is active and whether the induction gene is expressed by expressing the target gene in the activated and inactivated state of the CAR-T cell.

FIG. 6 shows IFN-gamma release in response to co-culture with nalm6 cells (see Table 6 below). Cells were cultured to day 8 and then NT cell levels were used to differentiate CAR ratio of CD19 CAR-T cells and CD19CAR-6xNFAT-GFP to CD19CAR-6xNFAT-IL6-2a-ifnγ cells. Will 10 ⁴ Car+ cells and 10 ⁴ Nalm-6 cells were co-cultured or cultured separately. After 24 hours, the supernatant was collected and the amount of IFN-gamma released was measured. Cells were co-cultured with nalm6 cells and in an activated state. Thus, the NFAT element initiates transcription of IFN-gamma in the activated state, thereby allowing release of IFN-gamma. When CAR-T cells are co-cultured with target cells, the cells are activated because the CAR-T cells recognize membrane proteins on the surface of tumor cells. The NFAT element initiates transcriptional translation of genes expressed as a result of activation. Expressing the corresponding expressed gene. When the cell is at rest or has a low level of activation, the NFAT element does not initiate transcriptional translation of the gene of interest in an inactive state. Thus, it can be judged by expressing the target gene in the activated and inactivated state of the CAR-T cellWhether NFAT elements are active or not and whether the inducible gene is expressed or not.

Table 6

Figure 7 shows toxicity assays for CAR T cells. T cells from healthy donors were cultured to day 8 and then NT cell levels were used to differentiate CAR ratio of CD19 CAR-T cells and CD19CAR-6xNFAT-GFP to CD19CAR-6xNFAT-IL6-2a-ifnγ cells. Will be 30X 10 ⁴ CAR+ cells were associated with 10 ⁴ Nalm-6 cells were co-cultured with 90e4 Nalm-6 cells. Nalm6 cell remnants were detected after 24 hours. CD19CAR-T cells, CD19CAR-6xNFAT-GFP cells and CD19CAR-6xNFAT-IL6-2a-ifnγ cells were co-cultured with nalm6 cells in different ratios. There was no significant difference between the 3 cell groups. After the CD19CAR-T, CD19CAR-6xNFAT-GFP and CD19CAR-6xNFAT-IL6-2a-IFN gamma cells are activated by tumor, the T cells play a role in killing target cells and leading the target cells to die.

Figures 8 and 9 show other IFN- γ release in response to co-culture with nalm6 cells. On day 0, peripheral blood T cells were obtained from volunteers and used with Dynabeads at 1: 1. On day 1, cells were infected with lentiviral vectors. On day 2, the medium was changed. Day 7 flow cytometry assays were used to detect CAR expression and CAR copy number. 1204 represents h19CAR,6107 represents h19CAR-2A-IL12, wherein the cell continuously expresses IL-12. The CAR expression was adjusted to 17%. Will 10 ⁴ CAR positive cells and 10 ⁴ Nalm6 or Nalm6-PDL1 tumor cells were co-cultured in 24 well plates for 24 hours and the supernatants were assayed for IFN-gamma. 1204 has 42% CAR expression, 1.5 copies per CART cell, 6107 has 23% CAR expression, 0.94 copies per CART cell, and CARs are balanced to 17% for co-culture. As shown in the bar graph, the co-culture results indicate that Nalm6-PDL1 stimulated at 1230 produced about half of the IFN gamma produced by Nalm6 stimulation due to the inhibition of T cells by PDL 1. IFN-gamma release from 6107 reaches about 10 times 1230, demonstrating that CART-released IL-12 significantly promotes IFN-gamma release.

FIGS. 10 and 11 showIL-12 and IFN-gamma release in response to CD3/CD28 Dynabeads activation. On day 0, peripheral blood T cells were obtained from volunteers and used with Dynabeads at 1: 1. On day 1, cells were infected with lentiviral vectors. On day 2, the medium was changed. On day 9, CAR expression was detected using flow cytometry. 1230 represents h19CAR and 6209 represents h19CAR-6xNFAT-IL12 (conditional release of IL-12 under T cell activation) to modulate CAR expression levels to 30%. Dynabeads at 1:3 stimulates the same number of cells for 24 hours and the supernatant is assayed for IL-12 and IFN- γ. There was 65% CAR expression in 1230 and 34% CAR expression in 6209. Dynabeads were added after leveling to 30%. After 24 hours, supernatant detection factor results were collected as a direct prescription at 6209 every 10 under the stimulation of Dynabeads ⁴ T cells release 55. Mu. gIL-12 on average. Regardless of the stimulus, 1230 does not release IL-12, and 6209 does not release IL-12 in the absence of the stimulus. This suggests that NFAT primordially activates IL-12 transcription under Dynabeads stimulation, as shown on the left of figure 11. As shown on the right in fig. 11, both CART cells did not release IFN- γ without stimulation, and 6209 released more IFN- γ with Dynabeads stimulation, indicating that IL-12 released more IFN- γ in concert with 6209CART cells.

FIGS. 12 and 13 show IL-6 and IFN-gamma release in response to CD3/CD28 Dynabeads activation. On day 0, peripheral blood T cells were obtained from volunteers and used with Dynabeads at 1: 1. On day 1, cells were infected with lentiviral vectors. On day 2, the media was changed. At day 7, CAR expression was detected using flow cytometry. 1230 represents h19CAR,6221 represents h19CAR-6xNFAT-IL6-2A-IFNγ (conditional release of IL6 and IFN- γ under T cell activation). The CAR expression was adjusted to 12.66%. Dynabeads at 1:3 stimulates the same number of cells for 24 hours and the supernatants are assayed for IL-6 and IFN-gamma. There was 67% CAR expression in 1230 and 29% CAR expression in 6221. Reach 12.66% and Dynabeads are added. After 24 hours, the supernatant test factor results were collected as a direct without Dynabeads and neither cell had IL-6 or IFN-gamma release. Upon addition of Dynabeads, 6221 significantly released more IL-6 and IFN- γ (per 10 ⁴ Average pg number of T cell release).

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

Table 7

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Claims (10)

1. A population of cells comprising an isolated nucleic acid sequence, the population of cells comprising: a nucleic acid sequence encoding a CAR that binds to an antigen of a leukocyte; an additional nucleic acid sequence encoding a therapeutic agent comprising IL-12; and a third nucleic acid sequence encoding an additional CAR that binds to a solid tumor antigen.

2. The population of claim 1, wherein the nucleic acid sequence, the additional nucleic acid sequence, and the third nucleic acid sequence are in the same cell or in different cells.

3. The population of cells of claim 1, wherein the CAR and therapeutic IL-12 are produced in the form of a polyprotein; which is cleaved to produce the CAR and therapeutic IL-12 alone, and/or each constitutively expressed by the CAR and therapeutic IL-12.

4. The population of cells of claim 3, said polyprotein comprising a cleavable moiety between the CAR and the therapeutic IL-12, the cleavable moiety comprising a 2A peptide, the 2A peptide comprising P2A or T2A.

5. A cell comprising an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a CAR that binds an antigen of a white blood cell and an additional nucleic acid sequence; the additional nucleic acid sequences encoding a therapeutic agent comprising IL-12.

6. The cell of claim 5, wherein the isolated nucleic acid sequence comprises SEQ ID NO 27.

7. The cell of claim 5 or 6, wherein the cell comprises: a third nucleic acid sequence encoding a further CAR different from the CAR, the further CAR binding to a solid tumor antigen.

8. A modified T cell, the T cell comprising a CAR, wherein the T cell comprises a nucleic acid sequence comprising: (1) a nucleic acid sequence encoding IL-6; and (2) a nucleic acid sequence encoding IFN-gamma; the T cells are engineered to express and secrete IL-6 and INF- γ when the T cells are activated, wherein the CAR comprises an extracellular domain, a transmembrane domain, an intracellular domain, an extracellular domain binding antigen, the CAR binding to a solid tumor antigen.

9. The T cell of claim 8, comprising a nucleotide sequence encoding SEQ ID NO:21 and 22, and SEQ ID NO:27 such that IL-6 and INF-gamma are expressed and secreted when T cells are activated.

10. The cell population of any one of claims 1-4, or the cell of claim 7, or the modified T cell of claim 8 or 9, wherein the solid tumor antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, qrr, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4a12, ALPP, CEA, ephA2, FAP, GPC3, IL13-rα2, mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR- α, 2, epCAM, EGFRvIII, PSCA, or EGFR.

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