CN110819596A - Modified cells with enhanced migratory capacity - Google Patents

Abstract

Embodiments of the present invention relate to modified T cells comprising an antigen binding molecule, wherein expression and/or function of CDC42 is enhanced in the modified cells. In embodiments, the modified cellular antigen binding molecule has an increased level of cytokine release in response to the antigen to which it binds compared to a corresponding T cell that does not overexpress CDC 42. In embodiments, cytokine release comprises release of IFN γ cytokines. In embodiments, the modified cell has enhanced migratory capacity for chemokines compared to a corresponding T cell that does not overexpress CDC 42.

Description

Modified cells with enhanced migratory capacity

Technical Field

The present disclosure relates to compositions and methods relating to chimeric antigen receptor cells and their use in treating diseases including cancer.

Background

Cancer, known as malignant tumor, involves abnormal cell growth and may invade or spread to other parts of the body. In humans, there are over one hundred cancers. Often, once cancer cells are shed, they spread throughout the body through the blood and/or lymphatic system, thereby endangering life. Currently, CAR-T therapy appears to be ineffective in treating solid cancers. One of the challenges is that CAR-T cells appear to be unable to migrate efficiently to these cancer cells that spread into the body.

Disclosure of Invention

Embodiments relate to T cells comprising an antigen binding molecule modification, wherein expression and/or function of CDC42 has been enhanced in the modified cell. In embodiments, the modified cell has an increased level of cytokine release in response to antigen bound by the antigen binding molecule as compared to a corresponding T cell that does not overexpress CDC 42. In embodiments, cytokine release includes cytokine release of IFN γ or GRAM B. In embodiments, the modified cell has enhanced migratory capacity for chemokines compared to a corresponding T cell that does not overexpress CDC 42.

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.

Drawings

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

Figure 1 shows an example of a CAR construct and an example of a CAR T cell;

figure 2 shows the results of a cytometric assay, indicating that CDC42 sequence does not affect expression of CARs on T cells;

figure 3 shows the results of a cytometric assay, indicating that CDC42 sequence does not affect the activation of CAR T cells;

figure 4 shows that CDC42 expression enhances GRAM B and IFN- γ release upon CAR activation;

figure 5 shows successful expression of CDC42 in CAR T cells;

figure 6 shows that the migration capacity of CDC42 expressing GUCY2C CAR T cells was significantly stronger than the control;

figure 7 shows the results of a cytometric analysis indicating that the ShRNA1 sequence does not affect expression of CARs on T cells, and also shows the results of a cytometric analysis indicating that the ShRNA1 sequence does not affect activation of CAR T cells;

FIG. 8 shows that ShRNA1 expression enhances the release of Gram B and IFN-. gamma..

Detailed Description

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 the purposes of this disclosure, the following terms are defined below.

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, "a component/element" means one component/element or more than one component/element.

"about" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

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

The term "antibody" is used in the broadest sense and refers to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodiesSex antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired biological activity or function. The antibodies of the present disclosure may exist in a variety of forms, including, for example, polyclonal, monoclonal, Fv, Fab' and F (ab)₂And fragments thereof; and single chain and humanized Antibodies (Harlow et al, 1999, In: Usingantibodies: 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-.

The term "antibody fragment" refers to a portion of a full-length antibody, such as an antigen-binding or variable region of an antibody. Other examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; 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 entire antigen recognition and antigen binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association. By folding of these two domains, six hypervariable loops (each 3 loops from the H and L chains, respectively) are created, 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 lower affinity than the entire binding site (dimer).

As used herein, "antibody heavy chain" refers to the larger of two types of polypeptide chains present in its naturally occurring conformation in all antibody molecules. As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains present in its naturally occurring conformation in all antibody molecules. The kappa and lambda light chains refer to the 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 a bacteriophage. The term also includes antibodies produced by synthesizing a DNA molecule encoding the antibody and expressing the DNA molecule to obtain the antibody or to obtain the amino acids encoding the antibody. Synthetic DNA is obtained using techniques available in the art and well known.

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, a DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response, thus encoding the term "antigen" as used herein. The antigen need not be encoded by only the full-length nucleotide sequence of the gene. The antigen may be generated, synthesized or derived from a biological sample, including a tissue sample, a tumor sample, a cell, or a biological fluid.

As used herein, the term "anti-tumor effect" refers to a biological effect associated with a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, a reduction in tumor cell proliferation, a reduction in tumor cell survival, an increase in the life expectancy of a subject having tumor cells, or an improvement in various physiological symptoms associated with a cancer condition. First, an "anti-tumor effect" can also be exhibited by the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis.

The term "autoantigen" refers to an endogenous antigen that is mistaken by the immune system for a foreign antigen. Autoantigens include cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

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

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

The term "xenogeneic" is used to describe grafts derived from subjects of different species. For example, the donor subject is from a different species than the 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 rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and "comprises" 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 means including and limited to anything following the phrase" consisting of. Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements may be present.

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

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

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

The term "co-stimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, TCR/CD3 complex binding to a peptide-loaded MHC molecule, which signal also mediates T cell responses, including at least one of proliferation, activation, differentiation, and other cellular responses the co-stimulatory ligand may include B7-1(CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, HLA-G, MICA, MICB, HVEM, lymphotoxin 40 receptor, 3/TR 40, ILT 40, lft 40, CD40, the binding of CD40, HLA-G, MICA, MICB, HVEM, lymphotoxin 40 receptor, antibody binding to a cell specific co-stimulatory ligand, CD40, and agonist binding to a cell specific ligand, e.g, CD40, and agonist binding to a cell-receptor of a cell-receptor, e.g-ligand, and a cell-ligand, such as a co-ligand specific for example, CD40, and a ligand binding to a ligand.

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

The term "co-stimulatory 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 key molecules. The terms "disease" and "condition" are used interchangeably and may also be different in that a particular disease or condition may not have a known pathogen (and therefore the cause has not been resolved) and therefore has not been considered a recognized disease, but merely an adverse condition or syndrome in which a clinician has identified a more or less set of particular symptoms. The term "disease" is a health state of a subject, wherein the subject is unable to maintain homeostasis, and wherein the health of the subject continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is less favorable than in the absence of the disease. If not treated in time, the condition does not necessarily lead to a further reduction in the health of the animal.

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

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

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

The term "expression or overexpression" refers to the transcription and/or translation of a particular nucleotide sequence into a precursor or mature protein, for example, driven by its promoter. By "overexpression" is meant the production of a gene product in a transgenic organism or cell that exceeds the production in a normal or non-transformed organism or cell. The term "expression" as defined herein refers to expression or overexpression.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control (regulatory) sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in vitro expression system. Expression vectors include all vectors 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) that incorporate recombinant polynucleotides.

The term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when a position in two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 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. When the two sequences are aligned, a comparison is made to obtain 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 major immunoglobulin produced in the primary immune response of most subjects, it is the most effective immunoglobulin in agglutination, complement fixation and other antibody reactions, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing mast cells and basophils to release mediators upon exposure to an allergen.

The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany it in its native state. The material may be a cell or a macromolecule, such as a protein or 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, e.g., a DNA fragment that has been removed from normal sequences that are normally adjacent to the fragment. Alternatively, "isolated peptide" or "isolated polypeptide" and the like, as used herein, refers to a peptide or polypeptide molecule that is isolated and/or purified in vitro from its native cellular environment and from its association with other components of a 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 instances, the term refers only to cells that have been isolated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

In the context of the present disclosure, the following abbreviations for ubiquitous nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanylic acid, "T" refers to thymine, and "U" refers to uridylic acid.

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

The term "lentivirus" refers to a genus of the family retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells. They can transmit a large amount of genetic information into the DNA of host cells, and thus they are one of the most effective methods in gene delivery vectors. In addition, the use of lentiviruses enables integration of genetic information into the host chromosome, thereby stably transducing the genetic information. HIV, SIV and FIV are examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

The term "modulate" refers to a detectable increase or decrease in the level of a response in a subject as compared to the level of a response in a subject in the absence of a treatment or compound, and/or as compared to the level of a response in a subject, and/or as compared to the level of a response in an otherwise identical, but untreated subject. The term includes interfering with and/or affecting 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 in a functional relationship with another polynucleotide. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, then the DNA is operably linked to the DNA for that polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

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

The term "overexpressed" 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, relative to the expression level in normal cells from the particular tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be identified by standard assays known in the art.

Cancers that may be treated include non-vascularized or not yet sufficiently vascularized tumors as well as vascularized tumors. Cancers may include non-solid tumors (e.g., hematological tumors, such as leukemia, lymphoma, and multiple myeloma), or may include solid tumors. The types of cancer treated with the disclosed CARs include, but are not limited to, carcinoma, blastoma, and sarcoma, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and pediatric tumors/cancers.

A hematologic cancer is a cancer of the blood or bone marrow. Examples of hematologic (or hematologic) cancers include leukemias, including acute leukemias (such as acute lymphocytic, acute myelocytic, acute myelogenous, and myeloblastic leukemias, promyelocytic leukemia, myelomonocytic, monocytic, and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous, and chronic lymphocytic leukemias), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphoma (both indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

A solid tumor is an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell type in which they are formed (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and central nervous system tumors (such as gliomas (such as brain stem glioma and mixed gliomas)), glioblastoma (also known as glioblastoma multiforme), astrocytoma, central nervous system lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

A 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. Table 1 provides examples of solid tumor antigens and their associated disease tumors.

TABLE 1

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 to refer to any human or animal suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human or an animal. In embodiments, the term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can 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 having a disease, condition, or disorder in need of treatment. Subjects in need thereof also include subjects in need of treatment to prevent a disease, condition, or disorder. In embodiments, the disease, condition, or disorder is cancer.

The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term generally refers to a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide that is 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 a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also include polynucleotides that differ from a 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 by 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 increased activity (i.e., is optimized) as compared to 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 therebetween, e.g., 90%, 95%, or 98%) sequence identity to a reference polynucleotide sequence described herein. The terms "polynucleotide variant" and "variant" 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 is a synthetic non-naturally occurring amino acid, e.g., a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides or "enzymes" that generally catalyze (i.e., increase the rate of various chemical reactions) a variety of 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, a polypeptide variant is distinguished from a 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 some amino acids may be changed to amino acids with broadly similar properties without changing the nature of the polypeptide activity. Polypeptide variants also include polypeptides in which one or more amino acids have been added or deleted or substituted with a different amino acid residue.

The term "promoter" refers to a DNA sequence that is recognized or introduced by the synthetic machinery of a cell and is required to initiate specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control (regulatory) sequences suitable for prokaryotes include, for example, promoters, optionally 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 adheres to a second molecule in a sample or organism, but does not substantially recognize or adhere 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 a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, this cross-species reactivity does not change the specificity of the antibody classification itself. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity does not change the specificity of the antibody classification itself. In some cases, the term "specifically binds" or "specifically binds" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on a chemical species; for example, antibodies recognize and bind to a specific protein structure rather than to any protein. If the antibody is specific for epitope "A", then in the reaction of labeled "A" and antibody, the presence of the epitope A-containing molecule (or free, unlabeled A) will reduce the amount of label A bound to the antibody.

By statistically significant, it is meant that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Common significance measures include p-value, i.e., the frequency or probability of an observation event occurring 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 0.05 or less. A "reduction" or "decrease" or "minor" amount is typically a "statistically significant" or physiologically significant amount, and can include 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 or more times (e.g., 100, 500, 1000 times) (including all decimal and integer numbers between 1 and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) reduction in the amounts or levels described herein.

The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, e.g., signaling via the TCR/CD3 complex.

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

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

The term "therapeutic" refers to treatment and/or prevention. Therapeutic effects can be obtained by inhibiting, reducing or eliminating the disease state or reducing the symptoms of the disease state.

The term "therapeutically effective amount" refers to the amount of a compound of the invention that will elicit the biological or medical response in a tissue, system or subject that is expected by a researcher, veterinarian, medical doctor or another clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more signs or symptoms of the disease or disorder being treated. A therapeutically effective amount will depend on the compound, the disease and its severity, as well as the age, weight and other factors of the subject to be treated.

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. The cells include primary test cells and progeny thereof.

The term "vector" refers to a polynucleotide that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to 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 an autonomously replicating plasmid or virus. 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 adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. For example, lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: and (6) SIV. Lentiviral vectors are created by multiple attenuation of HIV virulence genes, for example, by deletion of the genes env, vif, vpr, vpu and nef, rendering 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 in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have all the possible subranges specifically disclosed as well as individual numerical values within that range. For example, a description of a range 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 the stated range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth 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 or intracellular domain. In some embodiments, the domains of the CAR are on the same polypeptide chain, e.g., a chimeric fusion protein. In some embodiments, the domains are on different polypeptide chains, e.g., the domains are discontinuous.

The extracellular domain of the CAR molecule includes an antigen binding domain. In embodiments, the antigen binding domain binds to an antigen on the surface of a B cell, such as a cell surface molecule or marker. In embodiments, the cell surface molecule of a B cell comprises CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11B, CD18, CD169, CD1c, CD33, CD38, CD138, or CD 13. In embodiments, the cell surface molecule of a B cell is CD19, CD20, CD22, or BCMA. In a particular embodiment, the cell surface molecule of the B cell is CD 19.

Tumor antigens are well known in the art and include, for example, tumor-associated MUC1(tMUC1), glioma-associated antigens, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, α Alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), enterocarboxyesterase, muthsp70-2, M-CSF, protease, prostate-specific antigen (PSA), papap, nypap-ESO-1, LAGE-1a, p53, prostasin, PSMA, heru, telomerase, prostate cancer tumor antigen-1 (PCTA-1), machcar, escar-24, lag-1 a, p 8652, prostasin, PSMA, herr 2/neu, ghrin telomerase, prostate cancer tumor antigen-1 (PCTA-1), vacar, vacr 24, IGF-19, CD9, CD19, CD9, IGF-CD 19, CD 9.

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 linking the light and/or heavy chain variable regions using short linking peptides (Bird et al, Science242:423-426, 1988). An example of a linker peptide is a peptide having the amino acid sequence (GGGGS)₃(SEQ ID NO: 2) which bridges about 3.5nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al, 1988, supra). In general, the linker may be a short flexible polypeptide and preferably comprises about 20 amino acid residues or less. Single-stranded variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of an scFv, a suitable plasmid containing a polynucleotide encoding an scFv can be introduced into a suitable host cell, which can be a eukaryote, such as a yeast, plant, insect, or mammalian cell, or a prokaryote, such as e. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

In embodiments, the CAR molecules described herein comprise one or more CDRs for binding to a target antigen, e.g., one or more CDRs for binding to CD19 or tMUC 1.

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

In embodiments, the co-stimulatory domain comprises an intracellular domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, or any combination thereof. In embodiments, the signaling domain comprises a CD3 zeta domain derived from a T cell receptor.

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

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

Between the extracellular and transmembrane domains 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 link a transmembrane domain to an extracellular domain or a 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.

CAR molecules and therapeutic agents

The present disclosure describes a cell modified to express one or more molecules at a level that is higher or lower than the level of one or more molecules expressed by a cell that is not modified to express the one or more molecules, wherein the one or more molecules are associated with cell migration. Embodiments also describe modified cells modified to express an antigen binding molecule, wherein the expression and/or function of the one or more molecules in the modified cells, wherein the one or more molecules are associated with cell migration, has been increased or decreased. In some embodiments, the modified cell comprises a disruption of an endogenous gene or addition of an exogenous gene associated with a biosynthetic or transport pathway of the one or more molecules.

The terms "trafficking ability" and "migratory ability" of cells and the like are used interchangeably herein and refer to the ability of a cell (e.g., a T cell) to migrate in response to, for example, chemokines and stimuli (e.g., the tumor environment). Examples of chemokines can include CCL1, CCL5, CCL2, CCL22, CCL17, CXCL9, CXCL10, and CXCL 11. For example, modified T cells with CDC42 overexpression or reduced expression of LRCH1 show enhanced migratory capacity in migration assays. As shown in the examples below, the number of modified cells migrated or transported to the CCL5 containing medium was greater than the number of T cells that neither over-expressed CDC42 nor reduced LRCH1 expression.

The embodiments describe a method or use of a polynucleotide, the method comprising providing a viral particle (e.g., AAV, lentivirus or a variant thereof) comprising a vector genome, the vector genome comprising a polynucleotide encoding one or more molecules and a polynucleotide encoding a binding molecule, the polynucleotide operably linked to an expression control element to confer transcription of the polynucleotide; administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject, wherein the one or more molecules are associated with cell migration. In some embodiments, an AAV formulation can include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

Embodiments describe pharmaceutical compositions comprising a population of cells modified herein. Embodiments also describe a method of eliciting or 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 a pharmaceutical composition. The embodiments also describe isolated nucleic acid sequences encoding one or more molecules associated with cell migration.

In some embodiments, the one or more molecules comprise at least one of DOCK8, CDC42, and LRCH1, a functional variant of one or more molecules, or a functional fragment of one or more molecules. In some embodiments, the one or more molecules is or comprises CDC42, which is overexpressed in a modified T cell. In some embodiments, the one or more molecules is or comprises LRCH1, whose expression is reduced in modified T cells. In some embodiments, the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 and/or 39. In some embodiments, the modified cell comprises a polypeptide comprising SEQ ID NO: 38. In some embodiments, the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 and a recombinant polynucleotide encoding an antigen binding molecule (e.g., CAR). In some embodiments, the modified cell comprises a recombinant polynucleotide comprising the nucleotide sequence of SEQ ID NO: 38 and an encoded antigen binding molecule (e.g., CAR). In some embodiments, the modified cell comprises SEQ ID NO: 44-47.

A homologue of cell-division controlling protein 42, also known as Cdc42, is a protein involved in cell cycle regulation. It was originally identified in saccharomyces cerevisiae (yeast) as a mediator of cell division and is now known to affect a variety of signaling events and cellular processes in a variety of organisms from yeast to mammals. DOCK8 (cytokinesis inhibitor 8), also known as Zir3, is a large protein (about 190kDa) that participates in the intracellular signaling network, is a member of the guanine nucleotide exchange factors (GEFs) DOCK family of DOCK-C subfamilies, and serves as an activator of small G proteins. Leucine rich repeat and calpain homology domain containing protein 1(LRCH1) contains a leucine rich repeat and calpain homology domain and acts as a negative regulator of GTPaseCDC42, possibly by blocking CDC 42-guanine exchange factor DOCK8, possibly by blocking CDC42 activation, and negatively regulates CD4+ T cell migration.

In embodiments, the polynucleotide may be integrated into the genome of the modified cell, and progeny of the modified cell will also express the polynucleotide, thereby producing a stably transfected modified cell. In embodiments, the modified cell may express a polynucleotide encoding a CAR, but the polynucleotide is not integrated into the genome of the modified cell, such that the modified cell expresses the transiently transfected polynucleotide for a limited period of time (e.g., several days). The polynucleotide is then lost through cell division or other cellular processes. For example, the polynucleotide is present in the modified cell in the form of a recombinant DNA construct, mRNA or viral vector, and/or the polynucleotide is an mRNA that is not integrated into the genome of the modified cell.

Embodiments relate to methods or uses of the polynucleotides described herein. The method or use comprises: providing a viral particle (e.g., AAV, lentivirus or a variant thereof) comprising a vector genome comprising the polynucleotide, wherein the polynucleotide is operably linked to an expression control element that confers transcription of the polynucleotide; administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject. In an embodiment, the AAV formulation comprises AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. More information on the administration and preparation of viral particles can be found in U.S. patent nos.: 9840719, and sci. trans. med.11, eaav7325(2019), published by Milani et al, 5/2019, day 22, incorporated herein by reference.

In embodiments, cells with cell viability greater than 95% may be used at about 0.5 × 10⁶cells/mL density were seeded into the bioreactor. When the cell density reaches about 1.0X 10⁶cells/mL, cells can be transfected with Polyethyleneimine (PEI)/DNA complexes (multimers) in a ratio of 2: 1. at the time of harvest, AAV can be released from the cell culture in the bioreactor using the Triton X-100 method. All solutions can be added directly to the bioreactor and the lysate centrifuged at 4000 × g for 20 minutes. The supernatant can be stored at-80 ℃ for further processing. AAV may be further purified. For example, AAV samples (12.3mL) can be purified by overlaying the AAV sample with a series of iodixanol solutions with concentration gradients of 15%, 25%, 40%, and 54%, respectively, of 1, 5, 7, and 5 mL. A solution with 15% iodixanol concentration also contains 1M NaCl to avoid AAV aggregation with other cellular proteins and negatively charged nuclear components. After centrifugation is complete, 5mL of solution can be drawn 2 mm below the interface labeled 40/54 and then 3 in a Sorvals T-865 rotor in a Sorval ultracentrifugeUltracentrifugation at 85,000 Xg for 1 hour and 45 minutes. The viral vector can then be quantified. For example, in all cases, the infectivity of the vector AAV can be determined by Gene Transfer Assay (GTA) using GFP as a reporter gene. In AAV infectivity assays, samples are diluted to within 2% to 20% GFP-positive cells prior to their addition to the cells to ensure that only one virus enters the cells to express GFP. GFP positive cells can be quantified by FACS using HEK293 cells in suspension. AAV may then be administered to a subject. For example, AAV may be diluted in 0.9% sterile NaCl salt solution (supplemented with 0.25% human serum albumin [ HSA)]) To be infused into the patient, and the final infusion volume may be calculated at 3mL/kg depending on the weight of the patient.

In some embodiments, the modified cell comprises an antigen binding molecule and the antigen binding molecule is a CAR comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain in a cell, wherein the antigen binding domain that binds to a tumor antigen is selected from the group consisting of TSHR, CD, CD123, CD, CD171, CS-1, CLL-1, CD, EGFRvIII, GD, GD, BCMA, TnAg, PSMA, ROR, FLT, FAP, TAG, CD, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, mesothelin, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-beta, SSEA-4, CD, folate receptor (Her/neu), MUC, EGFR, NCAM, Prostase, Prostase, PFF 2, NHrin B, IGF-1 receptor, IGF-1, VLIX, VLAR 100, VLAR, CD receptor, CD11, CD 7H, CD7, CD, CD 7H, CD, CD, CD7, KIT, IL-13Ra, CD-I, CD-C, CD-11, CD-I, CD-G, CD-D, CD-G, CD-7-D, CD-G, CD-7, CD-G, CD-I, CD-G, CD-7-G, CD-D, CD-G, CD-G, CD-G, CD-G.

In some embodiments, the modified cell comprises an antigen binding molecule that is a modified TCR.

In some embodiments, the cell is an immune effector cell (e.g., a population of immune effector cells). In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. In some embodiments, the cell is a human cell.

In some embodiments, enhanced expression and/or function of one or more molecules is achieved by introducing nucleic acid sequences encoding the one or more molecules and/or binding molecules into modified cells in a recombinant DNA construct, mRNA, or viral vector. In some embodiments, the nucleic acid sequence is an mRNA that is not integrated into the genome of the modified cell. In some embodiments, the nucleic acid sequence is associated with an oxygen-sensitive polypeptide domain. In some embodiments, an oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain. In some embodiments, the nucleic acid sequence is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates expression of CDC42 in a cell. In some embodiments, the transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

In embodiments, the expression of one or more therapeutic agents may be modulated by an inducible expression system. Inducible expression systems allow for the temporally and spatially controlled activation and/or expression of genes. 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 a derivative thereof (e.g., doxycycline). For example, inducible suicide gene expression systems allow for the temporally and spatially controlled activation and/or expression of suicide genes, which results in cells killing themselves by apoptosis.

In embodiments, the modified cell comprises a nucleic acid sequence encoding a tetracycline transactivator (rtTA). In embodiments, the expression of one or more therapeutic agents is modulated by rtTA such that the one or more therapeutic agents are expressed in the presence of tetracycline. In embodiments, the concentration of tetracycline in the cell culture medium is not less than about 2 μ g/ml. In embodiments, the tetracycline is selected from tetracycline, demeclocycline, meclocycline, doxycycline, leimecycline, mecycline, minocycline, oxytetracycline, tetracycline, and chlortetracycline. In embodiments, the tetracycline is doxycycline.

In embodiments, the induced suicide system is an HSV-TK system or an induced caspase-9 system, in embodiments, the modified cell comprises a nucleic acid sequence encoding a suicide gene, such that the modified cell allows expression of the suicide gene in the presence of the nucleoside analog, rendering the nucleoside analog cytotoxic to the modified cell.

Embodiments relate to modified T cells comprising an antigen binding molecule, wherein expression and/or function of CDC42 in the modified cells has been enhanced. In embodiments, the modified cell has an enhanced T cell response. For example, T cell responses can be measured based on cytokine release levels, protease release levels, and/or migratory capacity. In embodiments, the modified cell has an increased level of cytokine release in response to antigen bound by the antigen binding molecule as compared to a corresponding T cell that does not overexpress CDC 42. In embodiments, the release of the cytokine comprises release of a cytokine of IFN γ. In embodiments, the protease release comprises release of granzyme b (gram b). Granzyme B is a serine protease, most commonly found in the granule of natural killer cells (NK cells) and cytotoxic T cells. It is secreted with the pore-forming protein perforin to mediate apoptosis of target cells. Recently, granzyme B has also been found to be produced by a variety of non-cytotoxic cells, from basophils and mast cells to smooth muscle cells. The accessory functions of granzyme B are also numerous. Granzyme B has been shown to induce inflammation by stimulating cytokine release and is also involved in the remodeling of the extracellular matrix. In embodiments, the modified cell has enhanced migratory capacity for chemokines compared to a corresponding T cell that does not overexpress CDC 42. In an embodiment, the chemokine is CCL 5. In embodiments, the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 and an antigen binding molecule. In embodiments, the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 in the form of a recombinant DNA construct, mRNA or viral vector in a modified cell. In embodiments, the polynucleotide is regulated by a promoter that includes a binding site for a transcriptional regulator that regulates expression of CDC42 in a cell. In embodiments, the transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB. Some embodiments relate to isolated nucleic acids comprising a polynucleotide.

The modified T cell may be derived from a stem cell. The stem cell may be an adult stem cell, an embryonic stem cell, more particularly a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell. The modified cell may also be a dendritic cell, an NK cell, a B cell or a T cell selected from the group comprising inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In another embodiment, the modified cell may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to expanding and genetically modifying the cells of the invention, the cells may be obtained from the subject by a variety of non-limiting methods. T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In embodiments, any number of T cell lines known to those of skill in the art may be used. In embodiments, the modified cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with infection. In embodiments, the modified cell is part of a mixed population of cells exhibiting different phenotypic characteristics.

The term "stem cell" refers to any of certain types of cells that have self-renewal ability and differentiate into other types of cells. For example, a stem cell will give rise to two daughter stem cells (e.g., embryonic stem cells cultured in vitro) or one stem cell and a blood cell that undergoes differentiation (e.g., a cell produced in a hematopoietic stem cell). Different classes of stem cells can be distinguished based on their source and/or their ability to differentiate into other types of cells. For example, stem cells may include Embryonic Stem (ES) cells (i.e., pluripotent stem cells), somatic stem cells, induced pluripotent stem cells, and any other type of stem cells.

Pluripotent embryonic stem cells may be present in the inner cell mass of blastocysts and have a high capacity for innate differentiation. For example, pluripotent embryonic stem cells may form any type of cell in vivo. ES cells maintain pluripotency when grown in vitro for long periods of time: progeny cells retain the potential for multilineage differentiation.

Somatic stem cells include fetal stem cells (from the fetus) and adult stem cells (present in various tissues, such as bone marrow). These cells are considered to have a lower differentiation potential than pluripotent ES cells, in which the differentiation potential of fetal stem cells is greater than that of adult stem cells. They apparently differentiate into a limited class of cells and are described as pluripotent. "tissue-specific" stem cells typically produce only one type of cell. For example, embryonic stem cells can differentiate into blood stem cells (e.g., Hematopoietic Stem Cells (HSCs)), which can further differentiate into various blood cells (e.g., red blood cells, platelets, white blood cells).

An induced pluripotent stem cell (i.e., iPS cell or iPSC) may include a pluripotent stem cell artificially derived from a non-pluripotent cell (e.g., adult somatic cell) by inducing expression of a specific gene. Induced pluripotent stem cells resemble natural pluripotent stem cells, such as Embryonic Stem (ES) cells, in many ways, such as expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling times, embryoid body formation, teratoma formation, feasible chimera formation, and potency and differentiability. Induced pluripotent cells can be made from adult stomach, liver, skin cells and blood cells.

In embodiments, the two different antigen binding domains may be on the same CAR and T Cell Receptor (TCR) and are encoded by separate nucleic acids.

Lymphocyte or T cell responses in a subject refer to cell-mediated immunity associated with helper, killer, regulatory and other types of T cells. For example, T cell responses may include activities such as assisting other White Blood Cells (WBCs) in the immune process and identifying and destroying virus infected cells and tumor cells. T cell responses in a subject can be measured by a variety of indicators, such as a number of virally infected cells and/or tumor cells that are killed by the T cells, the amount of cytokines released by the T cells when co-cultured with the virally infected cells and/or tumor cells, the level of proliferation of the T cells in the subject, such as a change in the phenotype of the T cells, a change in memory T cells, and the level of longevity or longevity of the T cells in the subject.

In embodiments, the method of enhancing a T cell response comprises treating a subject in need thereof, e.g., a subject diagnosed with a tumor. The term tumor refers to a tumor, which may be a collection of fluids such as blood, or a solid mass. Tumors can be malignant (cancerous) or benign. Examples of hematological cancers include chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphocytic leukemia, and multiple myeloma.

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

The five most common cancers include adrenal cancer, which involves organs that produce fluid or mucus, such as the breast and prostate. Basal cell carcinoma, cells involved in the outermost layer of the skin, such as skin cancer, squamous cell carcinoma, basal cells involved in the skin; transitional cell carcinoma affects transitional cells in the urinary tract, including the bladder, kidney, and ureter. Examples of the cancer include thyroid cancer, breast cancer, prostate cancer, lung cancer, intestinal cancer, skin cancer, pancreatic cancer, liver cancer, kidney cancer, and bladder cancer, and cholangiocarcinoma.

The methods described herein can be used to treat a subject diagnosed with cancer. The cancer may be a hematological cancer or may be a solid tumor, such as a sarcoma or carcinoma. The method of treatment includes 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 different from a cell surface molecule of White Blood Cells (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 the first antigen-binding domain and the second antigen-binding domain in vivo.

In embodiments, the first antigen binding domain is on a CAR and the second antigen binding domain is on a T Cell Receptor (TCR). In embodiments, the TCR is a modified TCR. In embodiments, the TCR is derived from a tumor-specific T cell that is spontaneously generated in the patient. In embodiments, the TCR binds a tumor antigen. In embodiments, the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1.

In embodiments, T cell clones expressing TCRs with high affinity for a target antigen can be isolated.A Tumor Infiltrating Lymphocyte (TIL) or PBMC can be cultured in the presence of Antigen Presenting Cells (APC) with an epitope-presenting polypeptide that represents an epitope that, when presented in the context of known HLA alleles, will elicit a dominant T cell response.A high affinity clone can then be selected based on the ability of the MHC-peptide tetramer to stain and/or recognize and lyse target cells with a low titer of cognate peptide antigen.after selection of the clone, TCR α and TCR β chains or the gamma and TCR delta chains are identified and isolated by molecular cloning.

Various methods can be performed to obtain a gene encoding a tumor-reactive TCR. More information is in Kershaw et al, Clin trans immunology.2014 5 months; 3(5): e 16. In embodiments, the specific TCR may be derived from a spontaneously generated tumor-specific T cell in the patient. Included within this class are antigens including the melanocyte differentiation antigens MART-1 and gp100, as well as the MAGE antigen and NY-ESO-1, which are expressed in a wider range of cancers. Whenever the transformed cells express viral proteins, TCRs specific for virus-associated malignancies can be isolated. Such malignancies include liver cancer and cervical cancer associated with hepatitis and papilloma virus, and malignancies associated with epstein-barr virus. In embodiments, target antigens for the TCR can include CEA (e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for melanoma), MAGE-A3 (e.g., for melanoma, esophageal and synovial sarcomas), NY-ESO-1 (e.g., for melanoma and sarcomas, and multiple myeloma).

In embodiments, the preparation and infusion of Tumor Infiltrating Lymphocytes (TILs) may be accomplished in the following manner. For example, tumor tissue from surgical or biopsy specimens can be obtained under sterile conditions and transported to a cell culture chamber placed in an ice box. Necrotic tissue and adipose tissue are removed. Tumor tissue is cut into pieces of about 1-3 cubic millimeters. Collagenase, hyaluronidase and DNase were added and digested overnight at 4 ℃. The cells were filtered through a 0.2 μm filter, and the cells were separated and collected by a lymphocyte separation medium (1500rpm, centrifugation for 5 minutes). Cells were expanded with medium containing PHA, 2-mercaptoethanol, and the CD3 monoclonal antibody, and a small dose of IL-2(10-20IU/ml) may be added to induce activation and proliferation. At 37 ℃ 5% CO₂Culturing for 7-14 days under the condition, detecting and maintaining the cell density at 0.5-2 × 10 according to growth condition⁶In the range of/ml. TIL positive cells have the ability to kill syngeneic cancer cells and can be screened by co-culture. Positive cells can be expanded in serum-free medium with high doses of IL2(5000-¹¹The TIL of (1). When administering TIL, it may be first collected in saline solution using continuous flow centrifugation and then filtered through a platelet administration device to a volume of 200 and 300ml containing 5% human serum albumin and 450000IU of IL-2. The TIL may be injected into the patient via a central venous catheter over a period of 30-60 minutes. In embodiments, the TILs may be injected into two to four separate bags; infusion may be several hours apart.

In embodiments, the isolated T cell comprises programmed death receptor 1(PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B-and T-lymphocyte attenuating agents (BTLA), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte activation protein 3(LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte associated immunoglobulin-like receptor 1(LAIR1), natural killer cell receptor 2B4(2B4), or a dominant-negative variant of CD 160. In embodiments, the isolated T cell comprises a reduced amount of TCR as compared to a corresponding wild-type T cell. The gene product of the dominant negative variant is altered, which antagonizes the wild-type allele. These mutations often result in altered molecular function (often inactive) and are characterized by a dominant or semi-dominant phenotype.

The present disclosure describes pharmaceutical compositions. The pharmaceutical composition comprises one or more of the following: CAR molecules, TCR molecules, modified CAR T cells, modified cells comprising a CAR or TCR, modified cells, nucleic acids, and vectors described herein. The pharmaceutical compositions are administered in a manner appropriate to the condition being treated or prevented. The appropriate dosage can be determined by clinical trials, and the amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease.

When an "immunologically effective amount", "an anti-tumor effective amount", "an effective tumor-inhibiting amount", or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician considering the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition (subject) of the patient. It can be said that a pharmaceutical composition comprising a T cell as described herein can be 10⁴To 10⁹Administration of a dose of individual cells/kg body weight, preferably 10⁵To 10⁶Individual cells per kg body weight, including all integer values within the range. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.Med.319: 1676, 1988). One skilled in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and adjusting the treatment accordingly. In certain embodiments, it may be desirable to administer activated T cells to a subject prior to drawing blood (or performing a singleton)Harvest), collecting the activated and expanded T cells, and injecting the patient with these activated and expanded T cells. This process may be performed multiple times every few weeks. In certain embodiments, T cells may be activated from 10cc to 400cc of blood draw. In certain embodiments, T cells are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of blood draw. Without being bound by theory, certain T cell populations may be selected using this multiple blood draw/multiple reinfusion protocol.

Administration of the pharmaceutical compositions described herein may be carried out in any convenient manner, including by inhalation by nebulization, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by intravenous (iv) injection or subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, transdermal (iv) injection. In embodiments, the T cell compositions described herein are administered to a subject by intradermal or subcutaneous injection. In embodiments, the T cell compositions of the invention are administered by intravenous injection. The composition of T cells may be injected directly into the tumor, lymph node or site of infection. In embodiments, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of related therapeutic methods, including but not limited to antiviral therapeutics, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab therapy for MS patients; efletuzumab therapy is used for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells described herein may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immune depleting agents (e.g., camp ath), anti-CD 3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, radiation, and the like. These drugs inhibit the calcium-dependent phosphatases calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin) important for growth factor-induced signaling. (Liu et al, Cell 66: 807-815, 1991; Henderson et al, Immun 73: 316-321, 1991; Bierer et al, curr. Opin. Immun 5: 763-773, 1993). In embodiments, the cell compositions described herein are administered to a subject in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation, T cell ablation therapy (using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH). In embodiments, the cell compositions described herein are administered to a patient after B cell ablation therapy (e.g., an agent that reacts with CD20, such as rituximab). In embodiments, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the invention. In embodiments, the expanded cells are administered before or after surgery.

In embodiments, CpG oligonucleotides (e.g., B class CpG oligonucleotides) can be systemically and repeatedly administered to a subject by introducing macrophage activation to enhance the anti-tumor effect of a drug described herein (e.g., CAR T cells). For example, CAR T cells and CpG oligonucleotides can be administered in combination to treat a subject having a solid tumor. Information on CpG oligonucleotide administration can be found in Nat immunol.2019 at 3 months; 20(3): 265-275, which is incorporated by reference in its entirety.

The above therapeutic dosages administered to a subject in need thereof will vary with the exact nature of the condition being treated and the recipient of the treatment. Scaling of the dose for human administration may be performed by a physician, depending on various factors and experience.

Additional information regarding methods of cancer treatment using engineered or modified T cells is provided in U.S. patent No. US8,906,682, which is incorporated by reference in its entirety.

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

The disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for the purpose of illustration 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 which become apparent as a result of the teachings provided herein.

Exemplary embodiments

The following are exemplary embodiments:

1. an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an antigen binding molecule and an additional nucleic acid sequence associated with disruption of an endogenous gene or addition of an exogenous gene of at least one of LRCH1, DOCK8, and Cdc 42.

2. A modified cell, comprising: an antigen binding molecule; and disruption of an endogenous gene or addition of an exogenous gene associated with a biosynthetic or transport pathway of at least one of LRCH1, DOCK8, and Cdc42, wherein the amount of DOCK8 or Cdc42 and/or the amount of LRCH1 is increased in the modified cell compared to a wild-type cell.

3. The modified cell of embodiment 2, wherein the endogenous gene associated with the biosynthesis or transport pathway of LRCH1 is disrupted in the modified T cell.

4. The modified cell of embodiment 3, further comprising a targeting nucleic acid sequence of SEQ ID NO: TALEN of 24.

5. The modified cell of embodiment 3, wherein the TALEN comprises a nucleic acid sequence comprising SEQ ID NO: 34 and a left arm comprising the nucleic acid SEQ ID NO: 35 right arm.

6. The modified cell of embodiment 3, further comprising a targeting nucleic acid sequence of SEQ ID NO: ZFN of 23.

7. The modified cell of embodiment 3, wherein the ZFN comprises a nucleic acid sequence comprising SEQ ID NO: 26-29 and a left arm comprising the nucleic acid SEQ ID NO: 30-33.

8. The modified cell of embodiment 2, wherein said DOCK8 or Cdc42 is overexpressed compared to a corresponding wild-type cell.

9. The modified cell of embodiment 2, wherein the expression level of DOCK8 or Cdc42 is at least about 10%, 20%, 30%, 40%, or 50% greater than the average expression level of cellular DOCK8 or Cdc 42.

10. The modified cell of embodiment 2, wherein the genome of the cell comprises a polynucleotide sequence encoding DOCK8 or Cdc42 operably linked to a promoter polynucleotide sequence.

11. The modified cell of embodiment 2, wherein said modification is a lymphocyte, a leukocyte or a PBMC.

12. A pharmaceutical composition comprising a population of cells according to any one of embodiments 2-11.

13. A method 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 the composition of embodiment 10.

14. The isolated nucleic acid sequence, modified cell, or method of any of embodiments 1-11, wherein the binding molecule is a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain binds an antigen.

15. The isolated nucleic acid sequence, modified cell or method of embodiment 12, wherein the intracellular domain comprises a costimulatory signaling domain comprising a sequence 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.

16. The isolated nucleic acid sequence, modified cell or method of embodiment 13, wherein the antigen is Epidermal Growth Factor Receptor (EGFR), variant iii of epidermal growth factor receptor (egfrviii), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bissialoganglioside 2(GD2), interleukin 13Ra2(IL13R α 2), Glypican-3(GPC3), carbonic anhydrase ix (caix), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG1B), mucin 1(MUC1), folate receptor- α (FR- α), CD19, FZD10, TSHR, PRLR, MUC 17, GUCY2C, CD3, CD 5B mature cell antigen (BCMA 4).

17. The isolated nucleic acid sequence, modified cell, or method of any of embodiments 2-14, wherein the modified cell comprises a dominant negative PD-1 mutant, thereby interfering with the PD-1/PD1-1 signaling pathway of the cell.

18. The isolated nucleic acid sequence, modified cell, or method of any of embodiments 2-15, wherein DOCK8 or Cdc42 is present in a recombinant DNA construct, mRNA, or viral vector in the modified cell.

19. The isolated nucleic acid sequence, modified cell, or method of any one of embodiments 2-15, wherein the modified cell comprises a DOCK8 or Cdc42mRNA encoding DOCK8 or Cdc42, and the mRNA is not integrated into the genome of the modified cell.

20. The isolated nucleic acid sequence, modified cell, or method of any of embodiments 2-19, wherein the modified cell comprises a nucleic acid sequence, or the isolated nucleic acid sequence comprises a promoter comprising a binding site for a transcriptional regulator that regulates expression of a therapeutic agent in the cell.

21. The isolated nucleic acid sequence, modified cell or method of embodiment 20, wherein said transcriptional modulator is or comprises Hif1a, NFAT, FOXP3 and/or NFkB.

22. The isolated nucleic acid sequence, modified cell or method of embodiment 21, wherein the promoter is responsive to a transcriptional regulator.

23. The isolated nucleic acid sequence, modified cell, or method of embodiment 22, wherein 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.

24. A cell modified to express one or more molecules at a level that is higher or lower than the level of the one or more molecules expressed by a cell that is not modified to express the one or more molecules, wherein the one or more molecules are associated with cell migration.

25. A modified cell engineered to express an antigen binding molecule in which the expression and/or function of one or more molecules is enhanced or reduced, wherein the one or more molecules are associated with cell migration.

26. The modified cell of any one of embodiments 24 and 25, wherein the modified cell comprises a disruption of an endogenous gene or addition of an exogenous gene associated with a biosynthetic or transport pathway of the one or more molecules.

27. A method or use of a polynucleotide, the method comprising:

providing a viral particle (e.g., AAV, lentivirus or a variant thereof) comprising a vector genome comprising a polynucleotide encoding one or more molecules operably linked to an expression control element that confers transcription of the polynucleotide and a polynucleotide encoding a binding molecule; and

administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject, wherein the one or more molecules are associated with cell migration.

28. The method of embodiment 27, wherein the AAV preparation may comprise AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

29. A pharmaceutical composition comprising a population of cells of any one of embodiments 24-26.

30. A method of eliciting or 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 the composition of embodiment 29.

31. An isolated nucleic acid sequence encoding one or more molecules associated with cell migration.

32. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 24-31, wherein said one or more molecules comprises one or more of DOCK8, CDC42 and LRCH1, a functional variant of said one or more molecules or a functional fragment of said one or more molecules.

33. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 24-32, wherein said one or more molecules is or comprises CDC42, said CDC42 being overexpressed in said modified T cell.

34. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 24-33, wherein the one or more molecules is or comprises LRCH1, the LRCH1 having reduced expression in a modified T cell.

35. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 24-34, wherein the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 and/or 39.

36. The modified cell of any of the preceding embodiments, wherein the modified cell comprises an antigen binding molecule and the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

37. The modified cell of embodiment 36, wherein said antigen binding domain binds to a tumor antigen selected from the group consisting of TSHR, CD, CD123, CD, CD, CD171, CS-1, CLL-1, CD, EGFRvIII, GD, GD, BCMA, TnAg, PSMA, ROR, FLT, FAP, TAG, CD, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, mesothelin, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-a, SSEA-4, CD, folate receptor (Her/neu), MUC, EGFR, NCAM, NYPAP, ELF2, Ephrin B, IGF-1 receptor, CAIX, LMP, gp100, bcr-abl, EphA, fucosylGM, GM, TGS, HMAA, NYO-acetyl receptor, WM/receptor, IGF-1 receptor, GARP, SALT-5, LACK-2, LACK-7, CAGE, MAG-7, CALT, MAG-7, CAGE, MAG-7, CALT, MAG-7, CAGE, MAG-7, CARD-LR-7, CARD-LR-7, CARD-LR-7, CAGE, CALL-7, CALL, MAG-S-7, MAG-LR-7, CALR-S, CALR-S, CARD-S, CALR-S, and CALR-S-7, CALR-S, CALR 2, CALR-S, CALRE 2, CALR-S, and CALR 2, and CALR 2.

38. The modified cell of any one of embodiments 36 and 37, wherein the intracellular signaling domain comprises a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of: CD, CD, 4-1BB (CD137), OX, CD, CD, PD-1, ICOS, lymphocyte function-associated antigen 1(LFA-1), CD, CD, LIGHT, NKG2, B-H, specifically binding CD, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), CD160, CD, CD, CD8alpha, CD8beta, IL2Rbeta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, DNAA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD RANGB, ITGB, CD, LyLFA-1, ITGB, TNFR, TRANCE/CD 226, ACAM, CD160, CD150, CD100, SLAMF-100, SLAMF-150, SLAM, SLA-6, CD49, CD11, ITGAE, CD103, ITGAL, CD11, CD100, CD-7, SELLPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG 2D.

39. The modified cell of any one of embodiments 24-30 and 31-35, wherein the modified cell comprises an antigen binding molecule and the antigen binding molecule is a modified TCR.

40. The modified cell of embodiment 39, wherein the TCR is derived from a spontaneously occurring tumor-specific T cell in the patient.

41. The modified cell of embodiment 40, wherein the TCR binds a tumor antigen.

42. The modified cell of embodiment 41, wherein said tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1.

43. The modified cell of embodiment 41, wherein the TCR comprises TCR γ and TCR δ chains or TCR α and TCR β chains, or a combination thereof.

44. The modified cell of any one of the preceding embodiments, wherein the cell is an immune effector cell. (e.g., immune effector cell populations).

45. The modified cell of embodiment 44, wherein the immune effector cell is a T cell or NK cell.

46. The modified cell of embodiment 45, wherein the immune effector cell is a T cell.

47. The modified cell of embodiment 45, wherein the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.

48. The modified cell of any one of the preceding embodiments, wherein the cell is a human cell.

49. The modified cell of any one of the preceding embodiments, wherein the enhancement of expression and/or function of the one or more molecules is achieved by introducing a nucleic acid sequence encoding the one or more molecules and/or binding molecules into the modified cell in a recombinant DNA construct, mRNA or viral vector.

50. The modified cell of embodiment 49, wherein the nucleic acid sequence is an mRNA that is not integrated into the genome of the modified cell.

51. The modified cell of embodiment 49, wherein the nucleic acid sequence is associated with an oxygen-sensitive polypeptide domain.

52. The modified cell of embodiment 51, wherein the oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain.

53. The modified cell of embodiment 49, wherein said nucleic acid sequence is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates expression of CDC42 in the cell.

54. The modified cell of embodiment 53, wherein said transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

55. A pharmaceutical composition comprising a population of cells of any one of embodiments 1-54.

56. A method of eliciting or 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 the composition of embodiment 55.

Examples of the present invention

Cells expressing chimeric receptors

Lentiviral vectors encoding a single CAR molecule were generated and used for T cell transfection, as will be described in detail below. The techniques associated with cell culture, the construction of cytotoxic T lymphocyte assays, can be found In "Control of large, established Molecular biology with genetic targeting human T cell conjugation CD28 and CD137 Domains," PNAS, March 3,2009, vol.106 No.9, 3360-. The corresponding sequences are shown in Table 2.

Table 2: sequences and identifiers

CART cell overexpression CDC42

PBMCs were obtained from volunteers and T cells were sorted on day 1. The vector containing GUCY2C CAR and the vector containing GUCY2C CAR-CDC42, respectively, were introduced into T cells (see table 3 for corresponding structures). Infected T cells were cultured until day 7. On day 7, 2X10⁵Individual T84 cells were seeded into 24-well plates. On day 8, 8X 10 cells were removed⁵GUCY2C CAR T cells and GUCY2C CAR-CDC42T cells were added to T84 coated 24-well plates. After 24 hours of co-cultivation, 3X 10 cells were obtained⁵Cells were phenotyped and supernatants were taken to determine release of GramB and IFN- γ. Phenotype was determined using flow cytometry. Figure 2 shows the results of the cell assay, indicating that CDC42 sequence does not affect the expression of CARs on T cells. Figure 3 shows the results of the cell assay, indicating that CDC42 sequence does not affect the activation of CAR T cells. Figure 4 shows that expression of CDC42 enhances release of GramB and IFN- γ upon CAR activation. The efficiency of overexpression of CDC42 was verified using qPCR. Figure 5 shows successful overexpression of CDC42 in CAR T cells. On day 7, 2X10 each⁶GUCY2C CAR T cells and GUCY2C CAR-CDC42 cells to verify the efficiency of overexpression of CDC 42. RNA was extracted using TRAzol, reverse transcription was performed using the RNA as a template, qPCR was performed using the reverse transcription as a template, and overexpression of CDC42 was measured.

TABLE 3

ID	Structure of the product
		GUCY2C	5F9-CD8hinge-41BB-CD3Z
GUC-CDC42	5F9-CD8hinge-41BB-CD3Z-P2A-CDC42
		GUCY2C	5F9-CD8hinge-41BB-CD3Z
GUC-SCR	U6-shRNA-Scremble-5F9-CD8hinge-41BB-CD3Z

Overexpression of CDC42 enhances the migratory capacity of CAR T cells

The effect of overexpression of CDC42 on cell migration ability was confirmed by the Transwell method. Day 9, 2X10⁵GUCY2C CAR T cells and GUCY2C CAR-CDC42 cells were placed in the upper plate of Transwell, respectively. 600 μ L of X-VIVO containing 50ng/mL CCL5 was placed in the Transwell lower plate. Flow cytometry assays were performed after 4 hours to measure the number of CAR + cells migrating in the underlying medium. Figure 6 shows that GUCY2C CAR T cells overexpressing CDC42 migrated significantly more than the control.

CAR T-cells overexpress LRCH1 and enhanced cytokine release

PBMCs were obtained from volunteers and T cells were sorted on day 1. The vector containing GUC-SCR and the vector containing ShRNA1 were introduced into T cells, respectively (see Table 3). Infected T cells were cultured until day 7. On day 7, 2X10⁵Individual T84 cells were seeded into 24-well plates. On day 8, 8X 10 of the above-mentioned herbs were sampled⁵GUC-SCR CAR T cells and ShRNA1CAR T cells and added to T84 coated 24-well plates. After 24 hours of co-cultivation, 3X 10 cells were obtained⁵Cells were phenotyped and supernatants were taken to determine release of GramB and IFN- γ. Phenotype was determined using flow cytometry. FIG. 7 shows the results of cellular assays demonstrating that the ShRNA1 sequence does not affect CAR at T-cellsExpression on a cell. The cellular assay results of figure 7 also indicate that the ShRNA1 sequence does not affect the activation of CAR T cells. FIG. 8 shows that ShRNA1 expression enhances release of GramB and IFN- γ upon CAR activation.

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.

Sequence listing

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Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp

180 185 190

Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu

195 200 205

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala Arg Gly Ser Arg Phe

210 215 220

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

225 230 235

<210>10

<211>244

<212>PRT

<213> scFv TSHR (Artificial sequence)

<400>10

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Ser Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Ala Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu

85 90 95

Gly Ile Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

115 120 125

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gln Ser Leu Lys

130 135 140

Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Asn Trp Ile Gly

145 150 155 160

Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile

165 170 175

Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln

180 185 190

Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr Leu Gln Trp

195 200 205

Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys Val Gly Leu

210 215 220

Asp Trp Asn Tyr Asn Pro Leu Arg Tyr Trp Gly Pro Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser

<210>11

<211>252

<212>PRT

<213> scFv PRLR (Artificial sequence)

<400>11

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Gly Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Gly

85 90 95

Glu Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

130 135 140

Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr

145 150 155 160

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

165 170 175

Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Ser Ser Leu Lys Ser GluAsp Ser Ala Met Tyr Tyr Cys

210 215 220

Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr Tyr Ala Met Asp

225 230 235 240

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

245 250

<210>12

<211>243

<212>PRT

<213> scFv Muc 17 (Artificial sequence)

<400>12

Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

1 5 10 15

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met Asn Trp Val Lys

145 150 155 160

Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr

165 170 175

Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu

180 185 190

Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Pro Tyr Tyr

210 215 220

Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210>13

<211>241

<212>PRT

<213> scFv GUCY2C (Artificial sequence)

<400>13

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Thr Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Arg

165 170 175

Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Gly Tyr Thr

210 215 220

Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser

<210>14

<211>246

<212>PRT

<213> scFv CD207 (Artificial sequence)

<400>14

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gln Val Gln Leu Arg Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

130 135 140

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp

145 150 155 160

Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp

165 170 175

Ile Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn

180 185 190

Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala

195 200 205

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

210 215 220

Cys Ala Thr Tyr Tyr Asn Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ala

245

<210>15

<211>199

<212>PRT

<213> Prolactin (ligand artificial sequence)

<400>15

Leu Pro Ile Cys Pro Gly Gly Ala Ala Arg Cys Gln Val Thr Leu Arg

1 5 10 15

Asp Leu Phe Asp Arg Ala Val Val Leu Ser His Tyr Ile His Asn Leu

20 25 30

Ser Ser Glu Met Phe Ser Glu Phe Asp Lys Arg Tyr Thr His Gly Arg

35 40 45

Gly Phe Ile Thr Lys Ala Ile Asn Ser Cys His Thr Ser Ser Leu Ala

50 55 60

Thr Pro Glu Asp Lys Glu Gln Ala Gln Gln Met Asn Gln Lys Asp Phe

65 70 75 80

Leu Ser Leu Ile Val Ser Ile Leu Arg Ser Trp Asn Glu Pro Leu Tyr

85 90 95

His Leu Val Thr Glu Val Arg Gly Met Gln Glu Ala Pro Glu Ala Ile

100 105 110

Leu Ser Lys Ala Val Glu Ile Glu Glu Gln Thr Lys Arg Leu Leu Glu

115 120 125

Gly Met Glu Leu Ile Val Ser Gln Val His Pro Glu Thr Lys Glu Asn

130 135 140

Glu Ile Tyr Pro Val Trp Ser Gly Leu Pro Ser Leu Gln Met Ala Asp

145 150 155 160

Glu Glu Ser Arg Leu Ser Ala Tyr Tyr Asn Leu Leu His Cys Leu Arg

165 170 175

Arg Asp Ser His Lys Ile Asp Asn Tyr Leu Lys Leu Leu Lys Cys Arg

180 185 190

Ile Ile His Asn Asn Asn Cys

195

<210>16

<211>240

<212>PRT

<213> scFv CD3 (Artificial sequence)

<400>16

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser Phe Ile Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

130 135 140

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

145 150 155 160

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

210 215 220

Ser Phe Ile Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

225 230 235 240

<210>17

<211>281

<212>PRT

<213> scFv CD4 (Artificial sequence)

<400>17

Met Asn Val Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Gly Gly Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser

20 25 30

Ala Ser Leu Glu Glu Ile Val Thr Ile Thr Cys Lys Ala Ser Gln Ala

35 40 45

Ile Asp Ala Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro

50 55 60

Gln Leu Leu Ile Tyr Asp Ala Thr Ser Leu Ala Asp Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser

85 90 95

Arg Pro Gln Val Asp Asp Ser Gly Ile Tyr Tyr Cys Leu Gln Ser Tyr

100 105 110

Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met

130 135 140

Ala Val Leu Val Leu Leu Leu Cys Leu Leu Ile Phe Pro Ser Cys Val

145 150 155 160

Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro

165 170 175

Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Thr

180 185 190

Ser Asn Ser Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Met Gly Val Ile Trp Ser Asn Gly Asp Ala Asp Tyr Asn Ser Ala

210 215 220

Ile Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val

225 230 235 240

Phe LeuLys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Met Tyr Phe

245 250 255

Cys Ala Ser Pro Tyr Tyr Gly Tyr Tyr Phe Pro Phe Asp Tyr Trp Gly

260 265 270

Gln Gly Val Met Val Thr Val Ser Ser

275 280

<210>18

<211>285

<212>PRT

<213> scFv CD4 (Artificial sequence)

<400>18

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Ser Ile Ser Ser His Asp Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Gln Pro Lys Leu Leu Ile Tyr Asp Ala Phe Asn Leu Ala Ser Gly

65 70 75 80

Ile Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Asp Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Tyr Cys Gln

100 105 110

Gln Ser Lys Asp Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu

115 120 125

Leu Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile

145 150 155 160

Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

165 170 175

Val Gln Pro Gly Arg Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe

180 185 190

Thr Phe Ser Asn Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys

195 200 205

Gly Leu Glu Trp Val Ala Thr Ile Ser Tyr Asp Gly Ser Ile Thr Tyr

210 215 220

Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala

225 230 235 240

Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr

245 250 255

Ala Thr Tyr Tyr Cys Thr Arg Glu Glu Gln Tyr Ser Ser Trp Tyr Phe

260 265 270

Asp Phe Trp Gly Pro Gly Ile Met Val Thr Val Ser Ser

275 280 285

<210>19

<211>242

<212>PRT

<213> scFv CD5 (Artificial sequence)

<400>19

Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu

115 120 125

Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys

130 135 140

Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp

145 150 155 160

Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp

165 170 175

Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr

180 185 190

Ile Thr Lys Asp Ala Ser Lys Asp Gln Val Ser Leu Lys Leu Ser Ser

195 200 205

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val Arg Arg Arg Ala

210 215 220

Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210>20

<211>244

<212>PRT

<213> ScFv MUC1-5e5 (Artificial sequence)

<400>20

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ile Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ser

130 135 140

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His

145 150 155 160

Ala Ile His Trp Val Lys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile

165 170 175

Gly His Phe Ser Pro Gly Asn Thr Asp Ile Lys Tyr Asn Asp Lys Phe

180 185 190

Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr

195 200 205

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

210 215 220

Lys Thr Ser Thr Phe Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

225 230 235 240

Thr Val Ser Ser

<210>21

<211>246

<212>PRT

<213> ScFv MUC1-Panko (Artificial sequence)

<400>21

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly Leu Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn

145 150 155 160

Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp

165 170 175

Val Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala

180 185 190

Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser

195 200 205

Ser Val Ser Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile

210 215 220

Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Thr Leu Thr Val Ser Ser

245

<210>22

<211>2291

<212>DNA

<213> LRCH1 (Artificial sequence)

<400>22

tggcgacgcc gggaagcgaa ccccaacctt tcgtcccggc cctttcggta gctactctgc 60

acccacttca tcatccccac caccaccacc accaccatca gcaccacgga ggaaccggcg 120

cccccggcgg ggcgggtggt ggcggcggtg gcagcggggg cttcaacctg cccttgaacc 180

ggggtctgga gcgcgcgctt gaggaggcgg ccaactccgg ggggctgaac ctgagcgcca 240

ggaaattgaa ggaatttccc cgtaccgcag cccccgggca cgacctctcg gacacggtgc 300

aggcagactt atctaaaaac agactggttg aagttccaat ggaattgtgc cattttgtat 360

cactggaaat tcttaatctg tatcacaact gtatcagagt cattcctgag gccatcgtta 420

atctgcagat gctgacttac ctgaacttga gtcgaaatca gctgtccgcc ctgcctgcct 480

gcctgtgtgg tctgcctctc aaagtcttaa tcgcaagtaa caacaaactt ggatcattac 540

cagaagagat aggtcagctc aaacagttaa tggagctgga tgtcagctgc aacgagatca 600

cagcgttgcc ccagcagata ggtcagttga aatctctacg agaactgaat gtcagaagaa 660

attaccttaa agttttacca caagaactag tagatcttcc cttggtaaag tttgactttt 720

cctgcaacaa agtgctcgtg attccaattt gttttagaga gatgaagcag ctgcaagtgt 780

tactacttga gaataaccct ctgcagtctc ctccagcaca gatttgcaca aagggcaaag 840

ttcacatatt taagtatctg agcatacaag catgccagat taagacagct gactcccttt 900

atctccacac catggagagg ccacatttac accagcacgt ggaagatggc aagaaggatt 960

ctgattcggg agttggaagt gataatggag ataagcgatt atctgccacc gagccttctg 1020

acgaagacac tgttagcctc aatgtgccaa tgtcaaacat catggaagaa gaacagatca 1080

tcaaggagga ctcgtgccat cgccttagcc ccgttaaagg ggaatttcat caggaatttc 1140

aaccggagcc ttcccttttg ggtgacagca ccaactcagg agaagaaaga gaccagttta 1200

ctgatagagc agatggtctc cattcggaat ttatgaacta taaggcaagg gcagaagact 1260

gtgaagagct gttacggata gaagaggatg tgcactggca aactgagggc ataataagtt 1320

catccaaaga tcaggacatg gatatagcaa tgatcgagca gctgagagaa gcagtagatt 1380

tgctgcaaga tcccaatgga ttaagcacag atattacaga gagaagtgtt ttaaacctat 1440

atcctatggg atcagcagaa gccttagaat tacaagattc tgcactgaat ggtcaaatac 1500

agctggagac atctccggtg tgtgaggtgc aaagtgatct aacattacag agtaacggga 1560

gccagtattc tccaaatgag attagagaga actcccctgc agtctctcct accacaaaca 1620

gcacagctcc atttggcctg aagcctcgat cagacccagc cctcattctt cctcctatct 1680

ccttcaacac acttacacag gcacagacat gggacagctc tagctacagt gttccatctg 1740

aaggagacag tgacaatgtg tttctaagac ctcagagaaa tttggaatct atagacccgc 1800

agtttacaat ccggaggaaa atggagcaga tgagagaaga gaaagagctg gtggaacaac 1860

ttcgtgagag cattgagatg agattgaagg tcagtctaca cgaagacctg ggggcagccc 1920

tcatggatgg tgtcgtcctc tgccatctgg tcaaccacat ccgcccacgg tcggttgcaa 1980

gcatccatgt cccatcacca gcggttccca aacttagcat ggccaaatgc agaagaaatg 2040

tggaaaactt tttggaagcg tgccgaaaat taggagtacc agaggctgac ctctgctctc 2100

cgtgtgacat cctgcagttg gattttcgtc acattcgaaa gactgttgac actctgctgg 2160

cactcgggga gaaagcccca ccaccaactt ctgccctccg ctccagggac cttataggct 2220

tctgtcttgt ccatattctc tttatagtgc tggtctatat cacttaccac tggaatgctc 2280

tgtccgcata a 2291

<210>23

<211>30

<212>DNA

<213> Target for ZFN (Artificial sequence)

<400>23

atggcgacgc cgggaagcga accccaacct 30

<210>24

<211>52

<212>DNA

<213> Target for Talen (Artificial sequence)

<400>24

tcgtcccggc cctttcggta gctactctgc acccacttca tcatccccac ca 52

<210>25

<211>23

<212>DNA

<213> Target for Crisper/Cas9 (Artificial sequence)

<400>25

tgaggaggcg gccaactccg ggg 23

<210>26

<211>7

<212>PRT

<213> ZFN Left F1 (Artificial sequence)

<400>26

Thr Ser Gly Asn Leu Thr Glu

1 5

<210>27

<211>7

<212>PRT

<213> ZFN Left F2 (Artificial sequence)

<400>27

His Thr Gly His Leu Leu Glu

1 5

<210>28

<211>7

<212>PRT

<213> ZFN Left F3 (Artificial sequence)

<400>28

Ser Arg Arg Thr Cys Arg Ala

1 5

<210>29

<211>7

<212>PRT

<213> ZFN Left F4 (Artificial sequence)

<400>29

Arg Ser Asp Lys Leu Thr Glu

1 5

<210>30

<211>7

<212>PRT

<213> ZFN Right F1 (Artificial sequence)

<400>30

Thr Lys Asn Ser Leu Thr Glu

1 5

<210>31

<211>7

<212>PRT

<213> ZFN Right F2 (Artificial sequence)

<400>31

Gln Ser Gly Asn Leu Thr Glu

1 5

<210>32

<211>7

<212>PRT

<213> ZFN Right F3 (Artificial sequence)

<400>32

Ser Lys Lys His Leu Ala Glu

1 5

<210>33

<211>7

<212>PRT

<213> ZFN Right F4 (Artificial sequence)

<400>33

Gln Ser Ser Asn Leu Val Arg

1 5

<210>34

<211>18

<212>DNA

<213> Left arm TALEN (Artificial sequence)

<400>34

tcgtcccggc cctttcgg 18

<210>35

<211>18

<212>DNA

<213> Right arm TALEN (Artificial sequence)

<400>35

acttcatcat ccccacca 18

<210>36

<211>16

<212>DNA

<213> Spacer (Artificial sequence)

<400>36

tagctactct gcaccc 16

<210>37

<211>48

<212>DNA

<213> Sh-RNA-SCREMBLE (Artificial sequence)

<400>37

gccggcagct agcgacgcca tctcgagatg gcgtcgctag ctgccggc 48

<210>38

<211>48

<212>DNA

<213> Lrch1-shRNA1 (Artificial sequence)

<400>38

gaccttatag gcttctgtct tctcgagaag acagaagcct ataaggtc 48

<210>39

<211>723

<212>DNA

<213> scFv GUCY 2C: 5F9 (Artificial sequence)

<400>39

gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ctccaggaga gagggccacc 60

ctgtcttgca gggctagcca gtccgtgtct aggaacctgg cctggtacca gcagaagcca 120

ggccaggctc ccagactgct gatctacgga gcttccacca gggctacagg aatcccagct 180

agattcagcg gctccggatc tggcaccgag tttaccctga caatcggctc tctgcagagc 240

gaggacttcg ccgtgtacta ctgccagcag tacaagacct ggcctcggac atttggacag 300

ggcacaaacg tggagatcaa gggaggagga ggatccggag gaggaggatc tggaggagga 360

ggaagccagg tgcagctgca gcagtgggga gctggcctgc tgaagccaag cgagacactg 420

tccctgacat gtgccgtgtt cggcggaagc ttttccggct actactggag ctggatcagg 480

cagccccctg gaaagggcct ggagtggatc ggagagatca accaccgggg caacaccaac 540

gacaacccct ccctgaagtc tcgcgtgacc atctctgtgg atacaagcaa gaaccagttc 600

gccctgaagc tgagctccgt gaccgctgct gacacagccg tgtactactg tgctagggag 660

agaggataca catacggcaa ctttgatcac tggggacagg gcaccctggt gacagtgtct 720

agc 723

<210>40

<211>22

<212>PRT

<213> P2A (Artificial sequence)

<400>40

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210>41

<211>69

<212>PRT

<213> CD8hinge (Artificial sequence)

<400>41

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210>42

<211>576

<212>DNA

<213> CDC42 Nucleotide (Artificial sequence)

<400>42

atgcagacca tcaagtgtgt ggtggtcgga gacggagccg tgggaaagac atgcctcctc 60

atctcctaca ccaccaacaa gttccccagc gagtacgtgc ccaccgtgtt tgacaactac 120

gccgtgaccg tgatgatcgg cggagagccc tacacactgg gactgttcga caccgccgga 180

caagaggatt acgatagact gagacctctg tcctatcctc agaccgatgt ctttctggtg 240

tgctttagcg tggtgagccc ctcctccttc gagaacgtga aagagaagtg ggtccccgag 300

atcacccacc actgccccaa aacccccttt ctgctggtgg gcacccagat tgatctgagg 360

gatgacccca gcaccatcga gaagctcgcc aagaacaagc agaagcccat tacccccgag 420

accgctgaga agctggctag agatctgaag gccgtcaagt acgtggagtg cagcgctctg 480

acccagaagg gactgaagaa tgtgttcgac gaagccattc tggccgctct ggagcctccc 540

gagcccaaaa agagcagaag gtgcgtgctg ctgtga 576

<210>43

<211>191

<212>PRT

<213> CDC42 aa (Artificial sequence)

<400>43

Met Gln Thr Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys

1 5 10 15

Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr

20 25 30

Val Pro Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly

35 40 45

Glu Pro Tyr Thr Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr

50 55 60

Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val

65 70 75 80

Cys Phe Ser Val Val Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys

85 90 95

Trp Val Pro Glu Ile Thr His His Cys Pro Lys Thr Pro Phe Leu Leu

100105 110

Val Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys

115 120 125

Leu Ala Lys Asn Lys Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys

130 135 140

Leu Ala Arg Asp Leu Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu

145 150 155 160

Thr Gln Lys Gly Leu Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala

165 170 175

Leu Glu Pro Pro Glu Pro Lys Lys Ser Arg Arg Cys Val Leu Leu *

180 185 190

<210>44

<211>2097

<212>DNA

<213>5F9-CD8hinge-41bb-CD3Z: Nucleotide (artificial sequence)

<400>44

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtcccctgcc acactgtccg tgtctccagg agagagggcc 120

accctgtctt gcagggctag ccagtccgtg tctaggaacc tggcctggta ccagcagaag 180

ccaggccagg ctcccagact gctgatctac ggagcttcca ccagggctac aggaatccca 240

gctagattca gcggctccgg atctggcacc gagtttaccc tgacaatcgg ctctctgcag 300

agcgaggact tcgccgtgta ctactgccag cagtacaaga cctggcctcg gacatttgga360

cagggcacaa acgtggagat caagggagga ggaggatccg gaggaggagg atctggagga 420

ggaggaagcc aggtgcagct gcagcagtgg ggagctggcc tgctgaagcc aagcgagaca 480

ctgtccctga catgtgccgt gttcggcgga agcttttccg gctactactg gagctggatc 540

aggcagcccc ctggaaaggg cctggagtgg atcggagaga tcaaccaccg gggcaacacc 600

aacgacaacc cctccctgaa gtctcgcgtg accatctctg tggatacaag caagaaccag 660

ttcgccctga agctgagctc cgtgaccgct gctgacacag ccgtgtacta ctgtgctagg 720

gagagaggat acacatacgg caactttgat cactggggac agggcaccct ggtgacagtg 780

tctagcacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag aggcgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgcggttc cggagccacg aacttctctc tgttaaagca agcaggagac 1500

gtggaagaaa accccggtcc tatgcagacc atcaagtgtg tggtggtcgg agacggagcc 1560

gtgggaaaga catgcctcct catctcctac accaccaaca agttccccag cgagtacgtg 1620

cccaccgtgt ttgacaacta cgccgtgacc gtgatgatcg gcggagagcc ctacacactg 1680

ggactgttcg acaccgccgg acaagaggat tacgatagac tgagacctct gtcctatcct 1740

cagaccgatg tctttctggt gtgctttagc gtggtgagcc cctcctcctt cgagaacgtg 1800

aaagagaagt gggtccccga gatcacccac cactgcccca aaaccccctt tctgctggtg 1860

ggcacccaga ttgatctgag ggatgacccc agcaccatcg agaagctcgc caagaacaag 1920

cagaagccca ttacccccga gaccgctgag aagctggcta gagatctgaa ggccgtcaag 1980

tacgtggagt gcagcgctct gacccagaag ggactgaaga atgtgttcga cgaagccatt 2040

ctggccgctc tggagcctcc cgagcccaaa aagagcagaa ggtgcgtgct gctgtga 2097

<210>45

<211>698

<212>PRT

<213>5F9-CD8hinge-41bb-CD3Z: aa (Artificial sequence)

<400>45

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys ArgAla Ser Gln

35 40 45

Ser Val Ser Arg Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile

85 90 95

Gly Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Lys Thr Trp Pro Arg Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr

165 170 175

Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Glu Ile Asn His Arg Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Glu Arg Gly Tyr Thr Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr

245 250 255

Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

305 310 315 320

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

325 330 335

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

340 345 350

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

370 375 380

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

385 390 395 400

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

405 410 415

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

420 425 430

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

435 440 445

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

450 455 460

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

465 470 475 480

Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

485 490 495

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gln Thr Ile Lys

500 505 510

Cys Val Val Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu Ile

515 520 525

Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr Val Pro Thr Val Phe

530 535 540

Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly Glu Pro Tyr Thr Leu

545 550 555 560

Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr Asp Arg Leu Arg Pro

565 570 575

Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val Cys Phe Ser Val Val

580 585 590

Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys Trp Val Pro Glu Ile

595 600 605

Thr His His Cys Pro Lys Thr Pro Phe Leu Leu Val Gly Thr Gln Ile

610 615 620

Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys Leu Ala Lys Asn Lys

625 630 635 640

Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys Leu Ala Arg Asp Leu

645 650 655

Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu Thr Gln Lys Gly Leu

660 665 670

Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala Leu Glu Pro Pro Glu

675 680 685

Pro Lys Lys Ser Arg Arg Cys Val Leu Leu *

690 695

<210>46

<211>1458

<212>DNA

<213>5F9-CD8hinge-41bb-CD3Z-P2A-CDC42 Nucleotide (artificial sequence)

<400>46

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtcccctgcc acactgtccg tgtctccagg agagagggcc 120

accctgtctt gcagggctag ccagtccgtg tctaggaacc tggcctggta ccagcagaag 180

ccaggccagg ctcccagact gctgatctac ggagcttcca ccagggctac aggaatccca 240

gctagattca gcggctccgg atctggcacc gagtttaccc tgacaatcgg ctctctgcag 300

agcgaggact tcgccgtgta ctactgccag cagtacaaga cctggcctcg gacatttgga 360

cagggcacaa acgtggagat caagggagga ggaggatccg gaggaggagg atctggagga 420

ggaggaagcc aggtgcagct gcagcagtgg ggagctggcc tgctgaagcc aagcgagaca 480

ctgtccctga catgtgccgt gttcggcgga agcttttccg gctactactg gagctggatc 540

aggcagcccc ctggaaaggg cctggagtgg atcggagaga tcaaccaccg gggcaacacc 600

aacgacaacc cctccctgaa gtctcgcgtg accatctctg tggatacaag caagaaccag 660

ttcgccctga agctgagctc cgtgaccgct gctgacacag ccgtgtacta ctgtgctagg 720

gagagaggat acacatacgg caactttgat cactggggac agggcaccct ggtgacagtg 780

tctagcacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag aggcgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgctaa 1458

<210>47

<211>485

<212>PRT

<213>5F9-CD5F9-CD8hinge-41bb-CD3Z-P2A-CDC 42: aa (Artificial sequence)

<400>47

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Arg Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile

85 90 95

Gly Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

100 105 110

Lys Thr Trp Pro Arg Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr

165 170 175

Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Glu Ile Asn His Arg Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Glu Arg Gly Tyr Thr Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr

245 250 255

Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

305 310 315 320

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

325 330 335

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

340 345 350

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

370 375 380

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

385 390 395 400

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

405 410 415

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

420 425 430

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

435 440 445

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

450 455 460

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

465 470 475 480

Ala Leu Pro Pro Arg *

485

<210>48

<211>723

<212>DNA

<213> scFv GUCY 2C: 5F9 (Artificial sequence)

<400>48

gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ctccaggaga gagggccacc 60

ctgtcttgca gggctagcca gtccgtgtct aggaacctgg cctggtacca gcagaagcca 120

ggccaggctc ccagactgct gatctacgga gcttccacca gggctacagg aatcccagct 180

agattcagcg gctccggatc tggcaccgag tttaccctga caatcggctc tctgcagagc 240

gaggacttcg ccgtgtacta ctgccagcag tacaagacct ggcctcggac atttggacag 300

ggcacaaacg tggagatcaa gggaggagga ggatccggag gaggaggatc tggaggagga 360

ggaagccagg tgcagctgca gcagtgggga gctggcctgc tgaagccaag cgagacactg 420

tccctgacat gtgccgtgtt cggcggaagc ttttccggct actactggag ctggatcagg 480

cagccccctg gaaagggcct ggagtggatc ggagagatca accaccgggg caacaccaac 540

gacaacccct ccctgaagtc tcgcgtgacc atctctgtgg atacaagcaa gaaccagttc 600

gccctgaagc tgagctccgt gaccgctgct gacacagccg tgtactactg tgctagggag 660

agaggataca catacggcaa ctttgatcac tggggacagg gcaccctggt gacagtgtct 720

agc 723

Claims (12)

1. A modified T cell comprising an antigen binding molecule, wherein expression and/or function of CDC42 is enhanced in said modified cell.

2. The modified T cell of claim 1, wherein the modified cellular antigen binding molecule has an increased level of cytokine release and/or protease release in response to the antigen to which it binds compared to a corresponding T cell that does not overexpress CDC 42.

3. The modified T cell of claim 2, wherein said cytokine release comprises release of IFN γ cytokines, wherein said protease release comprises release of GRAM B protease.

4. The modified T cell of claim 1, wherein the modified cell has enhanced migratory capacity for chemokines compared to a corresponding T cell that does not overexpress CDC 42.

5. The modified T cell of claim 4, wherein the chemokine is CCL 5.

6. The modified T cell of claim 1, wherein the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43 and the antigen binding molecule.

7. The modified T cell of claim 1, wherein the modified cell comprises an antigen binding molecule and the antigen binding molecule is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

8. The modified T cell of claim 7, wherein the antigen binding domain that binds to a tumor antigen is selected from the group consisting of TSHR, CD, CD123, CD, CD, CD171, CS-1, CLL-1, CD, EGFRvIII, GD, GD, BCMA, TnAg, PSMA, ROR, FLT, TRP, TAG, CD, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, mesothelin, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-beta, SSEA-4, CD, folate receptor (Her/neu), MUC, EGFR, NCRB, protease, PAP, ELF2, Ephrin B, IGF-1 receptor, CAIX, LMP, gp100, bcr-abl, EPDNAh, fucoidan, TGS, HMGA 11, CD-7, CD-I, CD-7, CD-1, CD-7, CD-1-, CD-1-, CD-, CD-and CD-1-, CD-OR CD-, CD-, CD-1-, CD-LR-, CD-7-, CD-and CD-7-, CD-, CD-7-, CD-1-, CD-7-, CD-OR CD-R-, CD-7-, CD-R-, CD-and CD-R-, CD-1-, CD-R-, CD-7-, CD-and CD-2-, CD-R-, CD-11-, CD-7-, CD-R-, CD-LR-, CD-R-, CD-7-, CD-R-, CD-1-, CD-7-, CD-OR CD-R-, CD-2-, CD-R-, CD-OR CD-7-, CD-2-, CD-R-, CD-7-, CD-R-, CD-2-, CD-7-, CD-R-, CD-7-, CD-R-.

9. The modified T cell of claim 1, wherein the modified cell comprises an antigen binding molecule and the antigen binding molecule is a TCR or a modified TCR.

10. The modified T cell of claim 1, wherein the modified cell comprises a nucleic acid sequence encoding SEQ ID NO: 43, and said polynucleotide is present in the modified cell in the form of a recombinant DNA construct, mRNA or viral vector.

11. The modified T cell of claim 10, wherein the polynucleotide is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates expression of CDC42 in a cell.

12. The modified T cell of claim 11, wherein the transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

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