Enhanced receptors to improve immune cell function

Abstract

The present invention provides an enhanced receptor that improves immune cell function, as well as related compositions and cells. The enhanced receptor is a transmembrane protein that constitutes a fusion protein consisting of both extracellular and intracellular domains, in which the extracellular domain can bind to target cells and initiate the function of the intracellular domain's signaling pathway, thereby increasing the activation level of immune cells, overcoming the suppressive effect of the target cell microenvironment on immune cells, and enhancing the efficacy of immunotherapy.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine technology, specifically to the field of immune cell therapy. [Background technology]

[0002] Conventional immunotherapy has limitations, primarily manifested as an insufficient level of immune cell activation. The microenvironment of target cells (such as cancer cells) exerts an inhibitory effect on immune cells, affecting their ability to kill target cells. Existing immune checkpoint monoclonal antibody drugs can partially alleviate the microenvironmental suppression, but their limitations are also evident. First, these antibodies indiscriminately suppress all T cells, even those that attack the body itself, posing a hidden risk of autoimmune diseases. Second, even if suppression is relieved, T cell function only recovers to a "normal" level, which is often insufficient to overcome advanced cancer. Summary of the Invention

[0003] In light of the above, there is a need for methods, systems, and products for improving immune cell function, and the present invention fulfills this need. The present invention provides an enhanced immune cell receptor, as well as related compositions and cells, which is a fusion protein composed of an extracellular domain (ECD) and an intracellular domain (ICD), in which the extracellular domain can bind to target cells and activate the function of the intracellular domain's signaling pathway, thereby increasing the activation level of immune cells, overcoming the suppressive effect of the target cell microenvironment on immune cells, and enhancing the efficacy of immunotherapy.

[0004] Unless otherwise indicated, the practice of the methods disclosed herein will employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (eds. F.M.A.usubel et al.), the series Methods in Enzyme Science (Academic Press, Inc.), PCR 2: A Practical Approach (eds. M.J. Machers, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane (eds. (1988)), Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (ed. R.I. Breshney (2010)).

[0005] The terms "about" and "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the measurement method or determination of the value, i.e., the extremes of the measurement system. For example, based on practice in the art, "about" can indicate within 1 or more standard deviations. Alternatively, "about" can indicate a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly in biological systems or processes, the term can indicate an order of magnitude, preferably within 5-fold, more preferably within 2-fold. When a particular value is recited in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0006] As used herein, "cell" typically refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of an organism. A cell may be from any organism having one or more cells. Non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, unicellular eukaryotic cells, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, bryophytes), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.). Cells include those derived from organisms other than humans (e.g., C. Agardh, etc.), seaweed (e.g., Laminaria japonica), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells derived from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells derived from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primary cells, humans, etc.), etc. Cells may not be derived from natural organisms (e.g., they may be synthetic, also known as artificial cells).

[0007] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent, such as an immune protein (e.g., an antibody). An antigen can also refer to a molecule or fragment thereof that is used to raise antibodies capable of binding to that antigen in an animal.

[0008] As used herein, the term "neoantigen" refers to a tumor-specific antigen, typically generated by genetic mutation. The resulting mutant protein or fragments thereof can elicit an anti-tumor T cell response.

[0009] As used herein, the term "gene" refers to a nucleotide sequence that encodes a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding RNA transcript. As used herein, the term with respect to genomic DNA includes intervening non-coding regions and regulatory regions, and may include the 5' and 3' ends. In some applications, the term includes the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some applications of the term, a "gene" includes only the coding sequence necessary to encode a polypeptide (e.g., an "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene can refer to a "foreign gene" or a non-native gene. A non-native gene can refer to a gene not normally found in a host organism but that is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location in the genome of an organism. Furthermore, a non-native gene can refer to a naturally occurring nucleic acid or polypeptide sequence, including mutations, insertions, and / or deletions (e.g., non-native sequences).

[0010] As used herein, the term "antibody" refers to a protein-binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) and derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Derivatives, variants, and fragments thereof can refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable region fragments (Fv), single-chain variable region fragments (scFv), microantibodies, diabodies, and single-domain antibodies ("sdAb" or "nanobody" or "camelid-derived"). The term antibody includes optimized, engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of antigen-engineered antibodies include Fc-optimized antibodies (eg, antibodies with optimized crystallizable fragment regions) and multispecific antibodies (eg, bispecific antibodies).

[0011] As used herein, the term "gene" refers to a nucleotide sequence that encodes a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding RNA transcript. As used herein, the term with respect to genomic DNA includes intervening non-coding regions and regulatory regions, and may include the 5' and 3' ends. In some applications, the term includes the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some applications of the term, a "gene" includes only the coding sequence necessary to encode a polypeptide (e.g., an "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene can refer to a "foreign gene" or a non-native gene. A non-native gene can refer to a gene not normally found in a host organism but that is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location in the genome of an organism. Furthermore, a non-native gene can refer to a naturally occurring nucleic acid or polypeptide sequence, including mutations, insertions, and / or deletions (e.g., non-native sequences).

[0012] As used herein, the term "antibody" refers to a protein-binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) and derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Derivatives, variants, and fragments thereof can refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable region fragments (Fv), single-chain variable region fragments (scFv), microantibodies, diabodies, and single-domain antibodies ("sdAb" or "nanobody" or "camelid-derived"). The term antibody includes optimized, engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of antigen-engineered antibodies include Fc-optimized antibodies (eg, antibodies with optimized crystallizable regions of the fragment) and multispecific antibodies (eg, bispecific antibodies).

[0013] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. A nucleotide can be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), and guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, and dTTP, or derivatives thereof. These derivatives can include, for example, (αS)dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled using well-known techniques. Labeling can also be performed with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0014] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and refer to a polymer of nucleotides, either deoxyribonucleotides or ribonucleotides, of any length, or analogs thereof, which may be in single-, double-, or multi-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases).

[0015] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template, for example, into mRNA or other RNA transcripts, and / or one or more processes by which the mRNA transcribed thereby is subsequently translated into peptides, polypeptides, or proteins. Transcription products and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. With respect to expression, "upregulation" typically refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to expression levels in the wild-type state, and "downregulation" typically refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to expression in the wild-type state.

[0016] As used herein, the term "modulation" with respect to expression or activity refers to changing the level of expression or activity. Modulation can occur at the transcriptional and / or translational level.

[0017] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymer of at least two amino acid residues linked by peptide bonds. The terms do not denote a specific length of the polymer, nor do they imply or distinguish whether the peptide is recombinantly, chemically, enzymatically, or naturally occurring. The terms apply to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms encompass amino acid chains of any length, including full-length proteins and proteins (e.g., domains) with or without secondary or tertiary structure. The terms also encompass amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation (e.g., conjugation with a labeling component). As used herein, the terms "amino acid" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and non-natural amino acids that have been chemically modified to include groups or chemical moieties not found in natural amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.

[0018] The terms "derivative," "variant," and "fragment," as used herein with respect to polypeptides, refer to polypeptides that are related to a wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Compared to the wild-type polypeptide, derivatives, variants, and fragments of a polypeptide can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof.

[0019] As used herein, "fusion" can refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., moieties). A fusion can include one or more identical non-native sequences. A fusion can include one or more different non-native sequences. A fusion can be chimeric. A fusion can include a nucleic acid affinity tag. A fusion can include a barcode. A fusion can include a peptide affinity tag. A fusion can provide subcellular localization of a site-specific polypeptide (e.g., a nuclear localization signal (NLS) for targeting the cell nucleus, a mitochondrial localization signal for targeting the mitochondria, a chloroplast localization signal for targeting the chloroplast, an endoplasmic reticulum (ER) retention signal, etc.). A fusion can provide a non-native sequence (e.g., an affinity tag) that can be used for tracking or purification. A fusion can be, for example, a small molecule such as biotin, or a dye such as Alexa fluor dye, Cyanine 3 dye, or Cyanine 5 dye.

[0020] As used herein, the phrase "artificial TCR" can be understood as a "foreign T cell receptor (TCR) complex," which refers to a TCR complex in which one or more chains of the TCR are introduced into the genome of an immune cell, which may or may not endogenously express a TCR. In some cases, an exogenous TCR complex can refer to a TCR complex in which one or more chains of the endogenous TCR complex have one or more mutated sequences, for example, at the nucleic acid or amino acid level. The expression of the exogenous TCR on an immune cell confers binding specificity for an epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of cancer cells or other disease-causing cells or particles). The exogenous TCR complex can contain a TCR-α, TCR-β chain, CD3-γ chain, CD3-δ chain, CD3-ζ chain, or any combination thereof, introduced into the genome. In some cases, the chains introduced into the genome can replace the endogenous chains.

[0021] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Additionally, they include tissues, cells, and the progeny thereof, of biological entities obtained in vivo or cultured in vitro.

[0022] As used herein, the terms "treatment" and "treating" refer to a method for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. For example, treatment can include administration of a system or cell population disclosed herein. A therapeutic benefit refers to any improvement or effect associated with treatment of one or more diseases, disorders, or symptoms under treatment. For a prophylactic benefit, the compositions can be administered to subjects at risk of developing a particular disease, disorder, or symptom, or to subjects who report one or more physiological symptoms of a disease, even if they may not yet be manifest. This is also true for diseases, disorders, or symptoms.

[0023] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, e.g., a composition comprising immune cells, such as lymphocytes (e.g., T lymphocytes and / or NK cells) of the present disclosure, sufficient to produce a desired activity when administered to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition that is sufficient to delay the onset of, prevent the progression of, alleviate, or alleviate at least one symptom of a disease treated by the methods disclosed herein.

[0024] As used herein, the term "genetic profile" refers to information about specific genes, including mutations and gene expression, in an individual or a type of tissue. Genetic profiles can be used for neoantigen selection. As used herein, the term "somatic mutation profile" refers to information about specific genes related to somatic mutations, including but not limited to specific genes produced by somatic mutations. Somatic mutation profiles can be used for neoantigen selection.

[0025] In one aspect, the present disclosure provides modified tumor-infiltrating lymphocytes (TILs) that specifically bind to tumor-associated antigens, including, but not limited to, neoantigens. The modified TILs may comprise chimeric stimulating molecules. The chimeric stimulating molecules may comprise a polypeptide extracellular domain (PED) that binds to the neoantigen. The PED can be fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal. Binding of the chimeric stimulating molecule to the neoantigen can result in an immune cell activation signal in the modified TILs. In some embodiments, the PED can be the extracellular domain of a surface protein of an unmodified TIL. In some embodiments, examples of PEDs include antibodies and derivatives, variants, and fragments thereof.

[0026] In one aspect, the present disclosure provides modified tumor-infiltrating lymphocytes (TILs) that specifically bind to neoantigens, the modified TILs comprising an enhanced receptor. The enhanced receptor can comprise the extracellular domain (ECD) of a protein. The ECD can be fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal. Binding of the enhanced receptor to a ligand can generate an immune cell activation signal in the modified TILs instead of an immune cell deactivation signal.

[0027] TILs can be any cells obtained from a tumor. For example, TILs can be cells that have migrated to a tumor. TILs can be cells that have infiltrated a tumor. In some embodiments, TILs are leukocytes that have migrated from a subject's bloodstream to a tumor. TILs can be, for example, T cells, B cells, monocytes, or natural killer (NK) cells. In some cases, engineered TILs include CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, or M1 macrophages. The immune cell population comprising TILs can also be a mixed cell population. TIL populations can include cells of different phenotypes, different degrees of differentiation, different lineages, or any combination thereof. TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect therapy. TILs can be classified based on the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD25, CD27, CD28, CD39, CD56, CD137, CCR7, CD45Ra, CD95, PD-1, and TIM-3. In some embodiments, the modified TILs express at least one of PD-1, CD39, CD137, and TIM-3. In some cases, TILs can be functionally defined by their ability to infiltrate solid tumors when reintroduced into a patient. In some cases, the modified TILs include "primary TILs," which refer to TILs obtained from a patient tissue sample. In some cases, the modified TILs include "secondary TILs," which refer to expanded or propagated TILs. TILs can express specific binding to neoantigens. In some cases, the TCR complex of the TILs confers antigen-binding specificity (e.g., neoantigen binding).

[0028] In one aspect, the present disclosure provides engineered T cells that specifically bind to neoantigens, the engineered T cells comprising an enhanced receptor. The enhanced receptor may comprise the extracellular domain (ECD) of a protein. The ECD can be fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal. Binding of the enhanced receptor to a ligand can generate an immune cell activation signal in the engineered T cells instead of an immune cell deactivation signal.

[0029] The engineered T cells can comprise a T cell receptor (TCR) complex that expresses specific binding to a neoantigen. In some embodiments, the TCR complex is an endogenous TCR complex. In some embodiments, the TCR is an exogenous TCR complex. The TCR complex (e.g., endogenous or exogenous) of the engineered immune cell can confer antigen-binding specificity (e.g., neoantigen binding) to the immune cell. In some embodiments, the present disclosure provides engineered T cells comprising an endogenous TCR complex that specifically binds to a neoantigen, the engineered T cells comprising a chimeric stimulating molecule, the chimeric stimulating molecule comprising a polypeptide extracellular domain (PED) that binds to a membrane protein on a cell, including but not limited to, a tumor cell, the PED fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and the chimeric stimulating molecule binds to the chimeric stimulating molecule. The membrane protein generates the immune cell activation signal in the engineered T cells.

[0030] Binding of engineered immune cells (e.g., engineered T cells or engineered TILs provided herein) to neoantigens can activate the immune cells. Enhanced receptors on engineered cells can provide additional control over immune cell activity, including, but not limited to, immune cell activation and proliferation. Binding of enhanced receptors to ligands in engineered immune cells (e.g., engineered T cells or engineered TILs) can induce immune cell activation signals in the engineered immune cells instead of immune cell deactivation signals. Inducing immune cell activation signals in the engineered immune cells instead of immune cell deactivation signals can minimize immunosuppressive effects in the immune cells. Minimizing immunosuppressive effects in immune cells can enhance the effectiveness of immune cells in an immune response, for example, by increasing immune cell cytotoxicity against target cells (e.g., tumor cells).

[0031] The enhanced receptor can include an extracellular domain (ECD) of a protein that induces an immune cell signal upon binding to a ligand in an unmodified immune cell. The signal can be an inactivating signal, an activating signal, or neither an activating nor inactivating signal. The protein can be a signaling receptor or any functional fragment, derivative, or variant thereof. In some cases, the signaling receptor can be a membrane-bound receptor. In response to ligand binding, the signaling receptor can induce one or more signaling pathways within the cell. In some cases, the signaling receptor can be a non-membrane-bound receptor. The enhanced receptor can include a fragment, e.g., an extracellular domain, of a receptor selected from a G protein-coupled receptor (GPCR), an integrin receptor, a cadherin receptor, an enzyme-linked receptor (e.g., a kinase), a death receptor, a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, a hormone receptor, and an immune receptor.

[0032] In some embodiments, the enhanced receptor comprises a fragment of an immune checkpoint receptor that can be involved in regulating the immune system. Non-limiting examples of such receptors include, but are not limited to, programmed cell death 1 (PD-1, or PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T-cell immunoglobulin mucin 3 (TIM-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), SIRPα, and NKG2D.

[0033] In some embodiments, the enhanced receptor comprises at least an extracellular fragment of a TCR, which may be involved in recognizing a neoantigen (e.g., a cancer cell antigen or a tumor antigen) on a target cell. In some instances, the enhanced receptor may comprise the extracellular variable region of the TCR alpha and / or beta chain.

[0034] The enhanced receptor can include an immune checkpoint receptor or any derivative, variant, or fragment thereof. The enhanced receptor can bind to an antigen comprising any suitable immune checkpoint receptor ligand or any derivative, variant, or fragment thereof. Non-limiting examples of such ligands include, but are not limited to, B7-1, B7-H3, B7-H4, HVEM (herpesvirus entry mediator), AP2M1, CD80, CD86, SHP-2, PPP2R5A, MHC (e.g., class I, class II), CD47, CD70, PD-L1 (or PDL1), and PD-L2. The region of the enhanced receptor that binds to such a ligand can be the natural receptor for such a ligand or a monoclonal antibody for such a ligand.

[0035] In some embodiments, the enhanced receptor comprises a fragment of a cytokine receptor. Cytokine receptors can perform many functions, non-limiting examples of which include regulating immune cells and mediating inflammation. In some embodiments, the enhanced receptor comprises a cytokine receptor, e.g., a type I cytokine receptor or a type II cytokine receptor, or any derivative, variant, or fragment thereof. In some embodiments, the enhanced receptor comprises an interleukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL-23R, IL-27R, and IL-31R), a colony-stimulating factor receptor (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR, and G-CSFR), a hormone receptor / neuropeptide receptor (e.g., growth hormone receptor, prolactin receptor, and leptin receptor), or any derivative, variant, or fragment thereof. In some embodiments, the enhanced receptor comprises a type II cytokine receptor, or any derivative, variant, or fragment thereof. In some embodiments, the enhanced receptor comprises an interferon receptor (e.g., IFNAR1, IFNAR2, and IFNGR), an interleukin receptor (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), a tissue factor receptor (also known as platelet tissue factor), or any derivative, variant, or fragment thereof.

[0036] In some embodiments, the extracellular binding domain of the enhanced receptor can be any antibody or antibody fragment, and the antigen to which the antibody binds can be any membrane protein that is widely expressed on human cells or that is expressed on tumor cells, including class I RTKs (e.g., the epidermal growth factor (EGF) receptor family, including EGFR, the ErbB family, including ErbB-2, ErbB-3, and ErbB-4), class II RTKs (e.g., the insulin receptor family, including INSR, IGF-1R, and IRR), class III RTKs (e.g., , the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), class IV RTKs (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), class V RTKs (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), class VI RTKs (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR / MET) and RON), class aClass VII RTKs (e.g., the tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB, and TRKC), class VIII RTKs (e.g., the ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), class IX RTKs (e.g., the AXL receptor family, such as AXL, MER, and TRYO3), class X RTKs (e.g., the LTK receptor family, such as LTK and ALK), class XI RTKs (e.g., the TIE receptor family, such as TIE and TEK), and class VIII RTKs (e.g., the TIE receptor family, such as TIE and TEK). Class XRTKs include, but are not limited to, Class XII RTKs (e.g., the ROR receptor family, such as ROR1 and ROR2), Class XIII RTKs (e.g., the discoidin domain receptor (DDR) family, such as DDR1 and DDR2), Class XIV RTKs (e.g., the RET receptor family, such as RET), Class XV RTKs (e.g., the KLG receptor family, which includes PTK7), Class XVI RTKs (e.g., the RYK receptor family, which includes Ryk), Class XVII RTKs (e.g., the MuSK receptor family, such as MuSK), CD47, CD70, NKG2D, or any derivative, variant, or fragment thereof.

[0037] In some embodiments, the enhanced receptor may comprise at least one extracellular region (e.g., a ligand-binding domain) of an enzyme-linked receptor (e.g., a receptor tyrosine kinase (RTK)) or any derivative, variant, or fragment thereof, or may comprise a fragment of an antibody variable region having these fragments as antigens. In some embodiments, the enhanced receptor is a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR, the ErbB family including ErbB-2, ErbB-3, and ErbB-4), a class II RTK (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), a class IV RTK (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), a class V RTK (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), a class VI RTK (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR / MET) and RON), a class II RTK (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), a class IV RTK (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), a class V RTK (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), a class VI RTK (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor Class VII RTKs (e.g., the tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB, and TRKC), Class VIII RTKs (e.g., the ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), Class IX RTKs (e.g., the AXL receptor family, such as AXL, MER, and TRYO3), Class X RTKs (e.g., the LTK receptor family, such as LTK and ALK), Class XI RTKs (e.g., the TIE receptor family, such as TIE and TEK), Class XII RTKs (e.g., ROR1 and ROR2 of the ROR receptor family), Class XIII RTKs (e.g., the discoidin domain receptor (DDR) family, such as DDR1 and DDR2), Class XIV RTKs (e.g., the discoidin domain receptor (DDR) family, such as DDR1 and DDR2), Class XIV RTKs (e.g., the AXL receptor family, such as AXL, MER, and TRYO3), Class XV RTKs (e.g., the LTK receptor family, such as LTK and ALK), Class XV RTKs (e.g., the TIE receptor family, such as TIE and TEK), Class XV RTKs (e.g., the ROR receptor family, such as ROR1 and ROR2), Class XVIII RTKs (e.g., the discoidin domain receptor (DDR)) family, such as DDR1 and DDR2), Class XVIII RTKs (e.g., the discoidin domain receptor (DDR)) family, such as DDR1 and DDR2These include RTKs (e.g., the RET receptor family, such as RET), Class XV RTKs (e.g., the KLG receptor family, which includes PTK7), Class XVI RTKs (e.g., the RYK receptor family, which includes Ryk), Class XVII RTKs (e.g., the MuSK receptor family, such as MuSK), CD47, CD70, NKG2D, or any derivative, variant, or fragment thereof.

[0038] Enhanced receptors include RTKs or any derivative, variant, or fragment thereof. Enhanced receptors can bind to any suitable RTK ligand or antigen, including any derivative, variant, or fragment thereof, or can comprise a fragment of an antibody variable region that targets these fragments as an antigen. Non-limiting examples of RTK ligands include growth factors, cytokines, and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-α or TGF-α, amphiregulin or AR, epiregulin or EPR, epigen, betacellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, neuregulin-4 or NRG4), fibroblast growth factor family (e.g., fibroblast growth factor family members, e ... FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21 and FGF23, etc.), vascular endothelial growth factor family (VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF, etc.), and platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC and PDGFD, etc.). Hormones include, for example, members of the insulin / IGF / relaxin family (e.g., insulin, insulin-like growth factor, relaxin family peptides including relaxin-1, relaxin-2, relaxin-3, Leydig cell-specific insulin-like peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5), and insulin-like peptide 6).

[0039] In some embodiments, the enhanced receptor comprises at least the extracellular region (e.g., ligand-binding domain) of an enzyme-linked receptor, such as a receptor threonine / serine kinase (RTSK), or any derivative, variant, or fragment thereof, or a fragment of the variable region of an antibody against these fragments. The enhanced receptor may comprise a type I RTSK, a type II RTSK, or any derivative, variant, or fragment thereof. The enhanced receptor may comprise a type I receptor selected from ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFβR1), ALK6 (BMPR1B), and ALK7 (ACVR1C), or any derivative, variant, or fragment thereof. The enhanced receptor may comprise a type II receptor selected from the group consisting of TGFβR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR), or any derivative, variant, or fragment thereof. In some embodiments, the enhanced receptor comprises a TGF-β receptor or any derivative, variant, or fragment thereof.

[0040] Enhanced receptors, including enhanced receptors for RTSK or any derivative, variant or fragment thereof, may bind to antigens including any suitable RTSK ligand or any derivative, variant or fragment thereof, or may comprise fragments of antibody variable regions against these fragments.

[0041] The enhancing receptor may include an intracellular domain (ICD) of a costimulatory molecule that induces an immune cell activation signal. The costimulatory molecule may bind to a ligand. In some cases, the costimulatory molecule may be activated by a ligand-responsive protein. In some embodiments, the costimulatory molecule is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the ICD is an intracellular domain of a costimulatory molecule and is selected from an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signal transduction molecule, a lymphocyte activation molecule (SLAM protein), an NK cell-activating receptor, BTLA, or a Toll ligand receptor. In some embodiments, the costimulatory molecule or costimulatory domain is selected from 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF. R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD3, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNF SF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACA M1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18,GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, IL-12R, integrin α4 / CD49d, integrin 4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SE LPLG(CD162), SLAM(SLAMF1), SLAM / CD150, SLAMF4(CD244), SLAMF6(NTB)-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF The signaling domain of a molecule selected from the group consisting of RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

[0042] The ECD and ICD of the enhanced receptor can be connected via a transmembrane domain, e.g., a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having a polypeptide containing at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution, deletion, or insertion compared to the transmembrane portion of the endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-naturally occurring polypeptide sequence, e.g., a polypeptide linker sequence. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the ECD, e.g., a signal indicating ligand binding, to the ICD. In some embodiments, the ECD comprises a transmembrane domain. In some embodiments, the ICD comprises a transmembrane domain.

[0043] In some embodiments, the ICD can mediate the production of an immune cell activation signal (also referred to as an immune cell excitation signal) in an immune cell. In some embodiments, the immune cell activation signal is mediated by an activator. The activator can be an immunomodulatory molecule. The activator can bind, activate, or stimulate T cells or other immune cells to regulate their activity. In some embodiments, the activator can be secreted from the immune cell. The activator can be, for example, a soluble cytokine, a soluble chemokine, or a growth factor molecule. Non-limiting examples of activators that can mediate immune cell activation include soluble cytokines, such as IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, tumor necrosis factor (TNF), transforming growth factor (TGF), interferon (IFN), or any functional fragment or variant thereof.

[0044] The immune cell activation signal may include or result in clonal expansion of the engineered immune cell (e.g., the engineered TIL or engineered T cell), cytokine release by the engineered immune cell (e.g., the engineered TIL or engineered T cell), cytotoxicity of the engineered immune cell (e.g., the engineered TIL or engineered T cell), proliferation of the engineered immune cell (e.g., the engineered TIL or engineered T cell), differentiation, dedifferentiation, or transdifferentiation of the engineered immune cell (e.g., the engineered TIL or engineered T cell), movement and / or trafficking of the engineered immune cell (e.g., the engineered TIL or engineered T cell), depletion and / or reactivation of the engineered immune cell (e.g., the engineered TIL or engineered T cell), release of other intercellular molecules, metabolites, chemical compounds, or a combination thereof, by the engineered immune cell (e.g., the engineered TIL or engineered T cell).

[0045] In some embodiments, the immune cell activation signal comprises or results in clonal expansion of the immune cell. Clonal expansion may comprise the production of daughter cells arising from the immune cell. The daughter cells resulting from clonal expansion may comprise the enhanced receptor. The clonal expansion of the modified immune cell may exceed the clonal expansion of a comparable immune cell lacking the enhanced receptor. The clonal expansion of the engineered immune cells is about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, or about 600-fold to about 700-fold compared to comparable immune cells lacking the enhanced receptor. In some embodiments, measuring clonal expansion can include, for example, quantitating the number of immune cells in the presence or absence of the enhanced receptor and after a ligand binds to the receptor. Quantitation of immune cell numbers is achieved by a variety of techniques, non-limiting examples of which include flow cytometry, trypan blue exclusion, and blood counts.

[0046] In some embodiments, the immune cell activation signal includes or results in cytokine release by the immune cell. In some embodiments, immune cell activity includes or results in the release of intracellular molecules, metabolites, chemical compounds, or combinations thereof. Cytokine release by the modified immune cell may include release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFNγ, TNFα, CSF, TGFβ, granzymes, etc. In some embodiments, cytokine release may be quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blot, etc. Cytokine release by the modified immune cell may exceed that of a comparable immune cell lacking the potentiating receptor. The modified immune cells provided herein can release approximately 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or 300-fold more cytokines than comparable immune cells lacking the enhanced receptor. When the enhanced receptor binds to a ligand and the modified immune cells bind to a neoantigen displayed on a target cell, the modified immune cells exhibit increased cytokine secretion compared to comparable (e.g., unmodified) immune cells lacking the enhanced receptor. In some embodiments, the secreted cytokine is IFNγ or IL-2. In some embodiments, cytokine release can be quantified in vitro or in vivo.

[0047] In some embodiments, the immune cell activation signal includes or results in the cytotoxic generation of immune cells. In some embodiments, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. An immune cell or population of immune cells expressing an enhanced receptor can induce the death of a target cell. Killing of target cells can have a variety of applications, including, but not limited to, the treatment of diseases or disorders in which a therapeutic removal of a population of cells or suppression of their proliferation is desired. Cytotoxicity can also refer to the release of cytotoxic cytokines, such as IFNγ and granzymes, by immune cells. In some cases, the modified immune cells provided herein can modify (i) the release of cytotoxins, such as perforin, granzymes, and granulysin, and / or (ii) the induction of apoptosis through Fas-Fas ligand interaction between T cells and target cells. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, including co-culture assays, ELISPOT assays, chromium-free cytotoxicity assays, and the like. The cytotoxicity of the modified immune cells provided herein can exceed the cytotoxicity of comparable immune cells lacking the enhanced receptor. The modified immune cells can exhibit increased cytotoxicity against target cells compared to comparable (e.g., unmodified) immune cells lacking the enhanced receptor when the enhanced receptor binds to its ligand and the modified immune cells bind to a neoantigen displayed on the target cell. The modified immune cells of the present disclosure can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more cytotoxic to target cells compared to comparable immune cells lacking the enhanced receptor. The modified immune cells of the present disclosure may induce death of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200% more target cells than comparable immune cells lacking the switch molecule.In some embodiments, the immune cells provided herein can induce apoptosis in target cells displaying a target epitope (e.g., a neoantigen) on their surface. In some embodiments, cytotoxicity can be measured in vitro or in vivo. In some embodiments, measuring cytotoxicity can include measuring the level of disease after administration of the modified immune cells provided herein compared to the level of disease before administration. In some embodiments, measuring cytotoxicity can include measuring the level of disease after administration of the modified immune cells provided herein compared to the level of disease after administration of comparable immune cells lacking the enhanced receptor.

[0048] In some embodiments, the immune cell activation signal comprises or results in immune cell proliferation. Immune cell proliferation can refer to the proliferation of a population of immune cells. Immune cell proliferation can refer to a change in the phenotype of an immune cell. The proliferation of modified immune cells of the present disclosure can exceed the proliferation of comparable immune cells lacking the potentiating receptor. The proliferation of the modified immune cells provided herein can exceed that of comparable immune cells lacking the potentiating receptor by about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, approximately 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, or about 600-fold to about 700-fold. In some embodiments, proliferation can be measured by quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue exclusion, and / or hemocytometry. Proliferation can also be determined by phenotypic analysis of immune cells.

[0049] In some embodiments, the immune cell activation signal may include or result in differentiation, dedifferentiation, or transdifferentiation of immune cells. Immune cell differentiation, dedifferentiation, or transdifferentiation can be determined by assessing the phenotypic expression of cell surface differentiation, dedifferentiation, or transdifferentiation markers by flow cytometry. In some embodiments, the modified immune cells provided herein have increased differentiation potential compared to comparable immune cells lacking the potentiating receptor. In some embodiments, the modified immune cells provided herein have increased dedifferentiation potential compared to comparable immune cells lacking the potentiating receptor. In some embodiments, the modified immune cells provided herein have increased dedifferentiation potential compared to comparable immune cells lacking the potentiating receptor. In some embodiments, the modified immune cells provided herein have greater transdifferentiation potential compared to comparable immune cells lacking the potentiating receptor.

[0050] In some embodiments, the immune cell activation signal may include or result in immune cell movement and / or trafficking. In some embodiments, movement may be measured by quantifying the localization of immune cells to a target site. For example, modified immune cells provided herein may be quantified at a target site, e.g., a non-target site, after administration. Quantification may be performed by isolating the lesion and quantifying the number of immune cells, e.g., tumor-infiltrating lymphocytes, that contain the enhanced receptor. The movement and / or trafficking of immune cells that contain the enhanced receptor may exceed the movement and / or trafficking of comparable immune cells that lack the enhanced receptor. In some embodiments, the number of immune cells that contain the enhanced receptor at a target site, e.g., a tumor lesion, may be about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, or 40-fold greater than the number of comparable immune cells that lack the enhanced receptor. Trafficking may also be measured in vitro using a transwell migration assay. In some embodiments, the number of immune cells comprising an enhanced receptor at a target site may be about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, or 40-fold greater than the number of equivalent immune cells lacking the enhanced receptor, e.g., in a transwell migration assay.

[0051] In some embodiments, the immune cell activation signal may include or result in the depletion and / or activation of immune cells. Immune cell depletion and / or activation may be measured by phenotypic analysis using flow cytometry or microscopy. Expression levels of depletion markers, such as programmed cell death protein 1 (PD1), lymphocyte activation gene 3 (LAG3), 2B4, CD160, Tim3, and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), may be measured quantitatively and / or qualitatively. In some cases, immune cells, such as T cells, may lose effector function and become depleted in a hierarchical manner. Depletion may result in loss of functions, such as IL-2 production and cytokine expression, and high proliferative capacity. Depletion may also lead to abnormalities in the production of IFNγ, TNF, and chemokines, as well as abnormalities in degranulation. The depletion or activation of the modified immune cells provided herein may exceed the depletion or activation of equivalent immune cells lacking the enhanced receptor. In some embodiments, the immune cells provided herein may provide at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or greater than 300-fold increase in depletion or activation compared to comparable immune cells lacking the potentiating receptor. In some embodiments, the immune cells provided herein may result in at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or greater than 300-fold decrease in depletion or activation compared to comparable immune cells lacking the potentiating receptor.

[0052] In some embodiments, binding of a target cell to the enhanced receptor can produce an immune cell activation signal in an immune cell that has been modified with the enhanced receptor.

[0053] In some embodiments, upon binding of the enhanced receptor to a ligand, the modified immune cell (e.g., modified TIL or modified T cell) with the enhanced receptor exhibits enhanced neoantigen binding compared to an immune cell lacking the enhanced receptor.

[0054] In some embodiments, immune cells (e.g., TILs, neoantigen-reactive T cells, CAR-Ts, TCR-Ts, NKs, etc.) engineered with an enhanced receptor can better overcome inhibitory signals from the tumor microenvironment to immune cells than immune cells engineered without the enhanced receptor (e.g., TILs, neoantigen-reactive T cells, CAR-Ts, TCR-Ts, NKs, etc.). The inhibitory signal may be derived from a ligand or antigen of the extracellular domain of the enhanced receptor, or may be an inhibitory signal from the tumor microenvironment to immune cells other than a ligand or antigen.

[0055] In some embodiments, the ECD of the potentiating receptor is an anti-PD1 or PDL1 monoclonal antibody, and the ICD is any type of costimulatory molecule, where the tumor-suppressing signals that can be overcome to a certain extent effectively may be not only from PDL1, but also immune cell-suppressing signals from CD47, TIM-3 ligands (such as Galectin-9), TIGIT ligands (such as CD155 and CD122 or PVR), or CTLA-4 ligands (such as B7).

[0056] In some embodiments, if the ECD of the potentiating receptor is anti-SIRPα or a CD47 monoclonal antibody, the ICD is any type of costimulatory molecule, and the tumor-suppressing signals that can be overcome to a certain extent effectively may be immune cell-suppressing signals not only from CD47, but also from PDL1, TIM-3 ligands (such as Galectin-9), TIGIT ligands (such as CD155 and CD122 or PVR), or CTLA-4 ligands (such as B77).

[0057] In some embodiments, when the ECD of the enhanced receptor is an anti-TIM-3 ligand monoclonal antibody (such as a Galectin-9 monoclonal antibody or an anti-TIM-3 monoclonal antibody) and the ICD is any type of costimulatory molecule, the tumor-suppressing signals that can be effectively overcome to a certain extent may be immune cell-suppressing signals not only from the anti-TIM-3 ligand (such as Galectin-9), but also from PDL1, TIGIT ligands (such as CD155 and CD122 or PVR), and CTLA-4 ligands (such as B77).

[0058] In some embodiments, when the ECD of the enhanced receptor is a monoclonal antibody of TIGIT or its ligand (such as an anti-CD155 monoclonal antibody, an anti-CD122 monoclonal antibody, or an anti-PVR monoclonal antibody), the ICD is any type of costimulatory molecule, and the tumor-suppressive signals that can be overcome to a certain extent effectively are not only from TIGIT ligands (such as CD155 and CD122 or PVR), but also immune cell-suppressive signals from PDL1, TIM-3 ligands (such as Galectin-9), or CTLA-4 ligands (such as B77).

[0059] In some embodiments, when the ECD of the enhanced receptor is VISTA, the ICD is any type of costimulatory molecule, and the tumor-suppressing signals that can be overcome to a certain extent effectively are not only from VISTA ligands, but also from CTLA-4 ligands (such as B7), and also from immune cell-suppressing signals from PDL1, CD47, TIM-3 ligands (such as Galectin-9), TIGIT ligands (such as CD155 and CD122 or PVR), or CTLA-4 ligands (such as B77).

[0060] In some embodiments, the T cells have a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and when the TCR or CAR can recognize target cells, the T cells modified with the enhanced receptor have a stronger immune cell activation function than T cells that have not been modified with the enhanced receptor; when neither the TCR nor the CAR can recognize target cells, the T cells modified with the enhanced receptor do not have a stronger immune cell activation function than T cells that have not been modified with the enhanced receptor.

[0061] In one aspect, the present disclosure provides engineered immune cells comprising a chimeric antigen receptor (CAR) and a T cell receptor (TCR) complex that expresses specific binding to a neoantigen. The CAR can include an antigen-interacting domain capable of binding to a B cell surface protein, a transmembrane domain, and an intracellular signaling domain.

[0062] The T cell receptor (TCR) complex that exhibits specific binding to the neoantigen may be an endogenous TCR complex or an exogenous TCR complex. The TCR complex (e.g., endogenous or exogenous) of the engineered immune cell can confer antigen-binding specificity (e.g., neoantigen binding) to the immune cell.

[0063] In some embodiments, the immune cells are tumor-infiltrating lymphocytes (TILs). TILs can be, for example, T cells, B cells, monocytes, or natural killer (NK) cells. In some cases, TILs include CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, or M1 macrophages. In some embodiments, TILs can express at least one of PD-1, CD137, and TIM-3. In some cases, the modified TILs include "primary TILs," which refers to TILs obtained from a patient tissue sample. In some cases, the modified TILs include "secondary TILs," which refers to expanded or propagated TILs. In some cases, TILs can reside in peripheral blood away from tumor tissue and can be obtained by isolating peripheral blood mononuclear cells (PBMCs) by apheresis, or further by screening or enrichment using one or more markers such as PD1, TIM3, CD137, CD39, etc.

[0064] CAR can comprise an antigen interaction domain that can bind to B cell surface protein. B cell surface protein can be any protein expressed on the surface of B cells. Non-limiting examples include CD1d, CD5, CD10, CD11a, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD29, CD34, CD37, CD38, CD40, CD44, CD45, CD49b, CD69, CD72, CD74, CD80, CD83, CD84, CD86, CD93, CD95, CD117, CD127, CD138, CD147, CD148, CD185, CD270, CD284 and CD360. In some embodiments, the antigen-interacting domain of the CAR can bind to a surface protein on a non-B cell, as long as binding to the surface protein does not significantly impair the general health or immune system of the host. In some embodiments, the surface protein is a surface protein on an immune cell. In some embodiments, the surface protein is a surface protein on a cell other than an immune cell. In some embodiments, the surface protein is selected from the group consisting of CD31, CD32, A, B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, a, b, c, d, CD43, CD44, CD45, CD46, CD47, CD48, CD49 (a, b, c, d, e, f), CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD61, CD62 (E, L, P), CD6 3, CD64 (A, B, C), CD66 (a, b, c), d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79 (a, b), CD80, CD81, CD82, CD83, CD84, CD85 (a, d, e, h, j, k), CD86 , CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD1(a~c), 1A, 1D, 1E, CD2, CD3(γ, δ, ε), CD4, CD5, CD6, CD7, CD8, a, CD9,CD10、CD11(a、b、c、d)、CD13、CD14、CD15、CD16、A、B、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD101、CD102、CD103、CD104、CD105、CD106、CD107(a、b)、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120(a、b)、CD121(a、b)、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD202b、CD204、CD205、CD206、CD207、CD208、CD209、CDw210(a、b)、CD212、CD213a(1、2)、CD217、CD218、(a、b)、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD233、CD234、CD235(a、b)、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD243、CD244、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD271、CD272、CD273、CD274、CD275、CD276、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD286、CD288、CD289、CD290、CD292、CDw293、CD294、CD295、CD297、CD298、CD299、CD300A、CD301、CD302、CD303、CD304、CD305、CD306、CD307, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD320, CD321, CD322, CD324, CD325, CD326, CD328, CD329, CD331, CD 332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD151, CD152, CD153, CD154, CD155, CD156 (a, b, c), CD157, CD158, (a, d, e, i, k), CD159(a, c), CD160, CD161, CD162, CD163, CD164, CD166, CD167(a, b), CD168, CD169, CD170, CD171, CD172(a, b, g), CD174, CD177, CD178, CD179(a, b), CD180, CD181, CD182, CD183, CD184, CD185 and CD186.

[0065] In some embodiments, the antigen-interacting domain of the CAR can bind to a B cell surface protein or a fragment thereof on a dead B cell. B cell apoptosis can occur before or after the onset of an immune response (e.g., an immune response to tumor cells). Thus, dead B cells or their debris can display a B cell surface protein or a fragment thereof on their surface. The ability of a CAR to target live and dead B cells can increase the chances that immune cells containing the CAR (i) bind to the B cell surface protein and (i) initiate signal transduction of the intracellular signaling domain. In some cases, signal transduction of the intracellular signaling domain can promote the proliferation (proliferation) of immune cells containing the CAR.

[0066] In some embodiments, the antigen-interacting domain of the CAR can bind to a B-cell surface protein or fragment thereof, which is attached (e.g., via covalent and / or non-covalent bonds) to the surface of a particle (e.g., a nanoparticle). The particle can be any particulate material, including organic and / or inorganic materials. The particle can have a variety of shapes and dimensions. The particle can be about 1 nanometer (nm) to about 50 nanometers (nm) in at least one dimension. The particle can be a minimum of about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm, or larger in at least one dimension. The particle can be a maximum of 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or smaller in at least one dimension. The particles may be nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanoribbons, etc. Exemplary particles include metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nanosemiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core with a polymer shell, particles with an organic core with a polymer shell, and mixtures thereof. Alternatively, the particles may be organic nanoparticles, such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxybutyric acid (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins), or combinations thereof. Particles displaying B cell surface proteins on their surfaces may be introduced in vitro into immune cells containing a CAR that binds to the B cell surface protein. Alternatively or additionally, particles displaying B cell surface proteins may be introduced in vivo (e.g., by local or systemic injection) along with CAR-containing immune cells. These particles can be used to expand populations of CAR-containing immune cells in vitro or in vivo.

[0067] The antigen-binding domain can comprise any protein or molecule capable of binding to an antigen, such as a B-cell surface protein. Non-limiting examples of antigen-binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, or functional derivatives, variants, and fragments thereof. They include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), microbodies, diabodies, and single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camelid-derived nanobodies. In some embodiments, the first antigen-binding domain comprises at least one of Fab, Fab', F(ab')2, Fv, and scFv. In some embodiments, the antigen-binding domain comprises an antibody mimetic. Antibody mimetics refer to molecules that can bind to target molecules with affinity comparable to that of antibodies, and include single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectins), lipocalin scaffolds, calixarene scaffolds, A-domains, and other scaffolds. In some embodiments, the antigen-binding domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the antigen-binding domain can comprise at least the ligand-binding domain of a transmembrane receptor.

[0068] In some embodiments, the antigen-interacting domain of the CAR can bind to a B cell surface protein or a fragment thereof on a dead B cell. B cell apoptosis can occur before or after the onset of an immune response (e.g., an immune response to tumor cells). Thus, dead B cells or their debris can display a B cell surface protein or a fragment thereof on their surface. The ability of a CAR to target live and dead B cells can increase the chances of immune cells containing the CAR (i) binding to the B cell surface protein and (ii) initiating signal transduction of the intracellular signaling domain. In some cases, signal transduction of the intracellular signaling domain can promote the proliferation (proliferation) of immune cells containing the CAR.

[0069] In some embodiments, the antigen-interacting domain of the CAR can bind to a B-cell surface protein or fragment thereof, which is attached (e.g., via covalent and / or non-covalent bonds) to the surface of a particle (e.g., a nanoparticle). The particle can be any particulate material, including organic and / or inorganic materials. The particle can have a variety of shapes and dimensions. The particle can be about 1 nanometer (nm) to about 50 nanometers (nm) in at least one dimension. The particle can be a minimum of about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm, or larger in at least one dimension. The particle can be a maximum of 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or smaller in at least one dimension. The particles may be nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanoribbons, etc. Exemplary particles include metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nanosemiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core with a polymer shell, particles with an organic core with a polymer shell, and mixtures thereof. Alternatively, the particles may be organic nanoparticles, such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxybutyric acid (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins), or combinations thereof. Particles displaying B cell surface proteins on their surfaces may be introduced in vitro into immune cells containing a CAR that binds to the B cell surface protein. Alternatively or additionally, particles displaying B cell surface proteins may be introduced in vivo (e.g., by local or systemic injection) along with CAR-containing immune cells. These particles can be used to expand populations of CAR-containing immune cells in vitro or in vivo.

[0070] The antigen-binding domain can comprise any protein or molecule capable of binding to an antigen, such as a B-cell surface protein. Non-limiting examples of antigen-binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, or functional derivatives, variants, and fragments thereof. They include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), microbodies, diabodies, and single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camelid-derived nanobodies. In some embodiments, the first antigen-binding domain comprises at least one of Fab, Fab', F(ab')2, Fv, and scFv. In some embodiments, the antigen-binding domain comprises an antibody mimetic. Antibody mimetics refer to molecules that can bind to target molecules with affinity comparable to that of antibodies, and include single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectins), lipocalin scaffolds, calixarene scaffolds, A-domains, and other scaffolds. In some embodiments, the antigen-binding domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the antigen-binding domain can comprise at least the ligand-binding domain of a transmembrane receptor.

[0071] In some embodiments, the antigen-binding domain may comprise an scFv. The scFv may be derived from an antibody with known variable region sequence. In some embodiments, the scFv may be derived from an antibody sequence obtained from an available mouse hybridoma. The scFv may be derived from the entire exon sequence of tumor cells or primary cells. In some embodiments, the scFv may be modified. For example, the scFv may be modified in various ways. In some cases, the scFv may be mutated to have higher affinity for its target. In some cases, the affinity of the scFv for its target may be optimized for a target that is expressed at low levels in normal tissues. This optimization may be performed to minimize potential toxicities such as hypercytokinemia. In other cases, cloning an scFv with higher affinity for a membrane-bound target may be preferable over its soluble counterpart. This modification may be performed when certain targets are detectable at different levels in soluble form and may cause unintended toxicities such as hypercytokinemia.

[0072] The antigen-binding domain of the CAR of the subject system is linked to the intracellular signaling domain via a transmembrane domain. The transmembrane domain can be a transmembrane segment. The transmembrane domain of the subject CAR can anchor the CAR to the plasma membrane of a cell, such as an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide linking the antigen-binding domain and the intracellular signaling domain of the CAR can have any suitable polypeptide. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having a polypeptide containing at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution, deletion, or insertion compared to the transmembrane portion of the endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-naturally occurring polypeptide sequence, such as a polypeptide linker sequence. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the extracellular region to the intracellular region of the cell via the antigen-binding domain. The natural transmembrane portion of CD28 can be used for CAR. In other cases, the natural transmembrane portion of CD8α can also be used for CAR.

[0073] The CAR of the present disclosure can include a signaling domain involved in immune cell signal transduction or any derivative, variant, or fragment thereof. The intracellular signaling domain of the CAR can induce the activity of immune cells containing the CAR. The intracellular signaling domain can transduce effector function signals to cause cells to perform specific functions. The signaling domain can include the signaling domain of other molecules. In some cases, a truncated portion of the signal region is used in the CAR.

[0074] In some embodiments, the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or any derivatives, variants, or fragments thereof. For example, the intracellular signaling domain can comprise at least two, e.g., at least 2, 3, 4, 5, 7, 8, 9, or 10 immune cell signaling domains. The immune cell signaling domains can be involved in regulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. The intracellular signaling domain can be the signaling domain of the T cell receptor (TCR) complex. The intracellular signaling domain of a subject CAR may comprise the signaling domain of Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain comprises an immunoreceptor tyrosine-dependent activation motif or ITAM. An ITAM-containing signaling domain can contain a dinucleotide repeat sequence of amino acids, YxxL, separated by 6-8 amino acids, where x is independently any amino acid, forming the conserved motif YxxL / Ix(6-8)YxxL / I. When the antigen-binding domain binds to an epitope, the ITAM-containing signaling domain can be modified, for example, by phosphorylation. The phosphorylated ITAM can serve as a docking site for other proteins, such as proteins involved in various signal transduction pathways.In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated, and / or optimized ITAM domain, which has modified (e.g., increased or decreased) activity compared to the native ITAM domain.

[0075] In some embodiments, the intracellular signaling domain of the subject CAR comprises an FcγR signaling domain (e.g., an ITAM). The FcγR signaling domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., an ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcαR signaling domain (e.g., an ITAM). The FcαR signaling domain may be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises an ITAM of CD3ζ.

[0076] In some embodiments, the intracellular signaling domain of the subject CAR comprises an immunoreceptor tyrosine-dependent inhibitory motif, or ITIM. Signaling domains containing ITIMs can include a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found in the cytoplasmic tails of several inhibitory receptors of the immune system. Key signaling domains containing ITIMs can be modified, e.g., phosphorylated, by enzymes such as members of the Src kinase family (e.g., Lck). Following phosphorylation, other proteins, including enzymes, can be recruited to the ITIM. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, the inositol phosphatases called SHIPs, and proteins with one or more SH2 domains (e.g., ZAP70). Intracellular signaling domains include BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), signaling domains of Fc receptors (e.g., ITIMs), similar protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), killer cell immunoglobulin (KCIM) receptor (KCR ... Killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor subfamily B member 3 (LIR3),Leukocyte immunoglobulin-like receptor subfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor subfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity-inducing receptor 2 (NKp44), NTB-A, programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid-binding Ig-like lectin 2 (SIGLEC2 or CD22), sialic acid-binding Ig-like lectin 3 (SIGLEC3 or CD33), sialic acid-binding Ig-like lectin 5 (SIGLEC3 or CD33), GLEC5 or CD170), sialic acid-binding Ig-like lectin 6 (SIGLEC6), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 10 (SIGLEC10), sialic acid-binding Ig-like lectin 11 (SIGLEC11), sialic acid-binding Ig-like lectin 4 (SIGLEC4), sialic acid-binding Ig-like lectin 8 (SIGLEC8), sialic acid-binding Ig-like lectin 9 (SIGLEC9), platelet / endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein 2 (SIRP2), and signal threshold regulating transmembrane adaptor 1 (SIT). In some embodiments, the intracellular signaling domain comprises a modified ITIM domain, e.g., a mutated, truncated, and / or optimized ITIM domain, which has modified (e.g., increased or decreased) activity compared to the native ITIM domain.

[0077] In some embodiments, the intracellular signaling domain comprises at least two ITAM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITAM domains). In some embodiments, the intracellular signaling domain comprises at least two ITIM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITIM domains) (e.g., at least two major signaling domains). In some embodiments, the intracellular signaling domain comprises both ITAM and ITIM domains.

[0078] In some cases, the intracellular signaling domain of the subject CAR can include a costimulatory domain. In some embodiments, a costimulatory domain, such as from a costimulatory molecule, can provide a costimulatory signal for immune cell signaling, such as signaling from an ITAM and / or ITIM domain, for example, for activation and / or inactivation of immune cell activity. In some embodiments, the costimulatory domain can be used to regulate proliferation and / or survival signals in immune cells. In some embodiments, the costimulatory signaling domain comprises an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), a signaling domain of activation, an NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, the costimulatory domain is 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, or B7. -H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100(SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD 300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49A, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26,DR3 / TNFRSF25, EPHB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros gene, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integron α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, LY9 (CD229), lymphoid Lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF The CAR comprises a signaling domain of a molecule selected from the group consisting of RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple costimulatory domains, e.g., at least two, e.g., at least three, four, or five costimulatory domains. The costimulatory signaling region can provide a signal that synergizes with the primary effector activation signal and can meet the requirements for T cell activation. In some embodiments, the addition of a costimulatory domain to the CAR can enhance the efficacy and persistence of the immune cells provided herein.

[0079] The binding of a CAR to a B cell surface protein can enhance the proliferation of immune cells compared to immune cells lacking a CAR. Proliferation of immune cells can refer to the proliferation of a population of immune cells. Proliferation of immune cells can refer to a change in the phenotype of immune cells. The proliferation of immune cells comprising a CAR provided herein can exceed the proliferation of immune cells lacking a CAR, and the immune cells express binding to a B cell surface protein. The proliferation of immune cells containing a CAR is about 5 to about 10 times, about 10 to about 20 times, about 20 to about 30 times, about 30 to about 40 times, about 40 to about 50 times, about 50 to about 60 times, about 60 to about 70 times, about 70 to about 80 times, about 80 to about 90 times, about 90 to about 100 times, about 100 to about 200 times, about 200 to about 300 times, about 300 to about 400 times, about 400 to about 500 times, about 500 to about 600 times, or about 600 to about 700 times greater than the proliferation of comparable immune cells lacking a CAR. The expansion of the CAR-containing immune cells is about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold greater than the expansion of comparable immune cells lacking the CAR, wherein the expansion is determined at least about 12, 24, 36, 48, 60, 72, 84, or 96 hours after contacting the B cells with the B cell surface protein. Enhanced proliferation is ensured both in vitro and in vivo. In some embodiments, proliferation can include quantification of immune cell numbers. Quantification of immune cell numbers can include flow cytometry, trypan blue exclusion, and / or hemocytometry. Proliferation can also be determined by immune cell phenotypic analysis.

[0080] In some aspects, the present disclosure provides engineered immune cells that specifically bind to a neoantigen, the engineered immune cells comprising: (a) a chimeric stimulating molecule comprising a polypeptide extracellular domain (PED) that binds to the neoantigen, where the PED is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the chimeric stimulating molecule to the neoantigen results in an immune cell activation signal in the engineered immune cell; and (b) a chimeric antigen receptor comprising (i) an antigen-interacting domain capable of binding to a B cell surface protein, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the PED can be the extracellular domain of a surface protein of an unmodified TIL. In some embodiments, examples of PEDs include antibodies and derivatives, variants, and fragments thereof.

[0081] In some embodiments, the present disclosure provides engineered immune cells that specifically bind to a neoantigen, wherein the engineered immune cells comprise: (a) a native TCR or a genetically engineered exogenous TCR capable of specifically binding to the neoantigen; (b) an enhanced receptor comprising an extracellular domain (ECD) of a protein, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the enhanced receptor to a ligand generates an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell; and (c) a chimeric antigen receptor comprising (i) an antigen-interacting domain capable of binding to a B cell surface protein, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein a is essential, and in some embodiments, only one of b or c may be present; and in some embodiments, a, b, and c coexist.

[0082] In one embodiment, the present disclosure provides an engineered tumor-infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, the engineered immune cell comprising: (a) a native TCR capable of specifically binding to the neoantigen; (b) an enhanced receptor comprising the extracellular domain (ECD) of a protein, where the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the switch molecule to its ligand can generate an immune cell activation signal in the engineered TIL instead of an immune cell inactivation signal; and (c) a chimeric antigen receptor comprising (i) an antigen-interacting domain capable of binding to a B cell surface protein, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. Of the three elements a, b, and c above, element a is essential; in some embodiments, only one of elements b or c may be present; and in some embodiments, elements a, b, and c coexist.

[0083] In one aspect, the present disclosure provides modified immune cells that overexpress a cytokine, e.g., a chemokine, where the immune cells are (i) tumor-infiltrating lymphocytes (TILs), (ii) stromal tumor-infiltrating lymphocytes (sTILs), or (iii) T cells that exhibit specific binding to an antigen. The modified immune cells that overexpress a chemokine can be any modified immune cell provided herein.

[0084] Cytokines refer to proteins (e.g., chemokines, interferons, lymphokines, interleukins, and tumor necrosis factors) released by cells that can affect cell behavior. Cytokines are produced by a variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by one or more cells. Cytokines can be involved in the production of systemic or local immunomodulatory effects.

[0085] Some cytokines can act as pro-inflammatory cytokines. Pro-inflammatory cytokines refer to cytokines involved in inducing or amplifying inflammatory responses. Pro-inflammatory cytokines can work with various cells of the immune system (e.g., neutrophils and leukocytes) to form an immune response. Some cytokines can function as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines involved in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can regulate the response of pro-inflammatory cytokines. Some cytokines can act as both pro-inflammatory and anti-inflammatory cytokines. Some cytokines, such as chemokines, can function in chemotaxis. Chemokines can induce directional chemotaxis in nearby responding cells.

[0086] In some embodiments, the expression of cytokines with pro-inflammatory and / or migratory activating functions can be upregulated in immune cells. Upregulation of the expression of cytokines with pro-inflammatory and / or migratory activating functions can be useful for stimulating an immune response against target cells, for example, in immunotherapy.

[0087] Examples of cytokines overexpressed by immune cells provided herein may include, but are not limited to, lymphokines, monokines, and traditional polypeptide hormones, including growth hormones (such as human growth hormone, N-methionine human growth hormone, and bovine growth hormone), parathormone, thyroxine, insulin, proinsulin, relaxant, prorelaxin, glycoprotein hormones (such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH)), hepatocyte growth factor, fibroblast growth factor, prolactin, placental prolactin, tumor necrosis factor-α, and Müllerian inhibitory factor. Factors, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF-α, etc.), platelet growth factor, transforming growth factors (TGFs, e.g., TGF-α, TGF-β, TGF-β1, TGF-β2, and TGF-β3), insulin-like growth factor-I and -II, erythropoietin (EPO), FLT-3L, stem cell factor (SCF), bone morphogenetic factor, interferon Interleukins (IFNs, e.g., IFN-α, IFN-β, IFN-γ), colony-stimulating factors (CSFs, e.g., macrophage-CSF (M-CSF)), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-stimulating factor (MSP), interleukins (ILs, e.g., IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10) , IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20), tumor necrosis factors (CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, etc.), and other polypeptide factors (including LIF, oncostatin M (OSM), and Kit ligand (KL)). Cytokine receptors refer to receptor proteins that bind cytokines. Cytokine receptors can be both membrane-bound and soluble.

[0088] In some embodiments, the overexpressed cytokine is a member of the interleukin (IL-1) family (e.g., a ligand), a member of the IL-1 receptor family, a member of the interleukin-6 (IL-6) family (e.g., a ligand), an IL-6 receptor, a member of the interleukin-10 (IL-10) family (e.g., a ligand), an IL-10 receptor, a member of the interleukin-12 (IL-12) family (e.g., a ligand), an IL-12 receptor, a member of the interleukin-17 (IL-17) family (e.g., a ligand), or an IL-17 receptor.

[0089] In some embodiments, the overexpressed cytokine is an interleukin-1 (IL-1) family member or related protein, a tumor necrosis factor (TNF) family member or related protein, an interferon (IFN) family member or related protein, an interleukin-6 (IL-6) family member or related protein, or a chemokine or related protein.In some embodiments, the cytokine is IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF / BLyS / TNFSF138, 4-1BBL, CD153 / CD30L / TNFSF8, CD40LG, CD70, Fas Ligand / FASLG / CD95L / CD178, EDA-A1, TNFSF14 / LIGHT / CD258, TNFA, LTA / TNFB / TN FSF1, LTB / TNFC, CD70 / CD27L / TNFSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-α / TNFA, TNFSF13, TL1 A / TNFSF15, OX-40L / TNFSF4 / CD252, CD40L / CD154 / TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, I FNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, Leptin, LIF, OSM, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, C CL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, C CL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4 and FAM19A5.

[0090] Various methods can be used to assess cytokine expression. Cytokine expression can be assessed for the presence of one or more cytokines by assaying cell culture medium in which the modified immune cells are cultured (e.g., in vitro production) or serum obtained from one or more sera obtained from a subject with the modified immune cells (e.g., in vivo production). Cytokine levels can be quantified using any suitable assay in various suitable units, including concentration. In some embodiments, cytokine protein is detected. In some embodiments, mRNA transcripts of the cytokine are detected. Examples of cytokine assays include enzyme-linked immunosorbent assays (ELISAs), Western blots, immunofluorescence assays, radioimmunoassays, antibody arrays that allow for parallel detection of various cytokines in a sample, bead-based arrays, quantitative PCR, microarrays, and the like. Other suitable methods can include proteomic techniques (e.g., two-dimensional gels, MS analysis, etc.).

[0091] In some embodiments, the cytokines overexpressed by the modified immune cells provided herein are chemokines. The chemokines can be, for example, CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines. In some embodiments, the chemokines overexpressed by the modified immune cells are CC chemokines selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28. The chemokine is a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17. In some embodiments, the chemokine overexpressed by the engineered immune cells is a C chemokine selected from XCL1 and XCL2. In some embodiments, the chemokine overexpressed by the immune cells is a CX3C chemokine, and the CX3C chemokine is CX3CL1.

[0092] In one aspect, the disclosure provides a method of treating cancer in a subject, comprising: (a) administering to the subject modified TILs, modified T cells that have been positively screened using one or more of PD1, TIM3, CD137, and CD39 in peripheral blood PBMCs, modified T cells, or modified immune cells of any one of various embodiments of the aspects described herein; and (b) contacting neoantigen-expressing cancer target cells with the modified TILs, modified and selected T cells, modified T cells, or immune cells under conditions that induce cytotoxicity of the modified TILs, modified and selected T cells, modified T cells, or modified immune cells that are modified against the target cells, thereby inducing death of the cancer target cells.

[0093] In some aspects, the disclosure provides methods of expanding a population of T cells, the methods including: (a) providing a population of T cells comprising at least one modified immune cell according to any one of various embodiments of the aspects described herein; and (b) exposing the population of T cells to a B cell surface protein to result in expansion of the T cell population. In some embodiments, in (b), the population of T cells is exposed to B cells comprising the B cell surface protein.

[0094] In one aspect, the present disclosure provides a method for expanding a population of T cells, comprising: (a) introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the population of T cells, thereby producing a cell or population of cells expressing the CAR, wherein the CAR comprises (i) an antigen-interacting domain that specifically recognizes the CAR, (ii) a transmembrane domain, and (iii) an intracellular signaling domain; and (b) contacting the population of CAR-expressing cells with an antigen, thereby producing a population of expanded and / or activated immune cells, wherein the antigen is not specifically expressed by tumor target cells. The antigen can be, for example, a B cell surface protein, such as CD19, BCMA, or other blood cell surface proteins, or a mutated version of a naturally occurring protein, such as EGFRviii, or an artificially engineered protein.

[0095] In one aspect, the present disclosure provides compositions comprising one or more polynucleotides encoding one or more of: (a) an enhanced receptor comprising the extracellular domain (ECD) of a protein fused to the intracellular domain (ICD) of a costimulatory protein that mediates an immune cell activation signal; and (b) an antigen-specific T cell receptor complex, or one or more components thereof.

[0096] In one aspect, the present disclosure provides compositions comprising one or more polynucleotides encoding one or more of: (a) an antigen-specific T cell receptor complex, or one or more components thereof; (b) a chimeric antigen receptor comprising: (i) an antigen-interacting domain capable of binding to a B cell surface protein, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

[0097] In certain aspects, the present disclosure provides compositions comprising one or more polynucleotides encoding one or more of: (a) an enhanced receptor comprising the extracellular domain (ECD) of a protein fused to the intracellular domain (ICD) of a costimulatory protein that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof; or (c) a chimeric antigen receptor comprising: (i) an antigen-interacting domain capable of binding to a B cell surface protein, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

[0098] In various embodiments of the aspects herein, promoters that can be used with the compositions of the present disclosure include promoters that are active in eukaryotic, mammalian, non-human mammalian, or human cells. The promoters can be inducible or constitutively active. Alternatively, or in addition, the promoters can be tissue- or cell-specific.

[0099] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include promoters derived from early cytomegalovirus (CMV), herpes simplex virus (HSV) thymidine kinase, early and late SV40, and long terminal repeats (LTRs). Retroviruses include promoters derived from the human elongation factor-1 promoter (EF1), hybrid constructs containing the cytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-1. The promoter may be a fungal promoter. The promoter may also be a plant promoter. Databases of plant promoters are well known (e.g., PlantProm). The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also contain appropriate sequences for amplifying expression.

[0100] In various embodiments of the present invention, engineered immune cells can specifically bind to neoantigens and / or neoepitopes. Neoantigens and neoepitopes generally refer to tumor-specific mutations, which in some cases trigger anti-tumor T cell responses. For example, these endogenous mutations can be identified using whole-exon sequencing. Tran E et al., "Cancer Immunotherapy in Epithelial Cancer Patients Based on Mutation-Specific CD4+ T Cells," Science 344:641-644 (2014). Engineered immune cells (e.g., engineered TILs or engineered T cells) containing enhanced receptors can exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by immune cells can be expressed on target cells and, for example, encoded by mutations in endogenous genes. In some cases, the neoantigens or neoepitopes specifically bound by immune cells can be encoded by mutant genes.The genes in question are ABL1, ACO1 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B (code NY-ESO-1), DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM111B, FGFR3, FRG1B, GAGE1, GAGE10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, and MAGEB. 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT. In some embodiments, neoantigens are selected based on the genetic profile of a tumor sample from an individual. In some embodiments, neoantigens are selected based on the somatic mutation profile of a tumor sample from an individual.

[0101] In various embodiments of the aspects herein, the modified immune cells further comprise an inactivation switch (or suicide switch). In cases where the injury is severe, such as in hypercytokinemia, the kill switch can be activated to eliminate the immune cells. This can occur when the immune system has such a strong response that many inflammatory cytokines are released, causing mild to severe symptoms including fever, headache, rash, increased heart rate, decreased blood pressure, and difficulty breathing. The death switch can be a drug-inducible death switch. The death switch can include inducible caspase 9.

[0102] Various embodiments of the present invention include cells, such as modified immune cells. Cells, such as immune cells (e.g., lymphocytes, including T cells and NK cells), can be obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and genetically modified species thereof. Examples of samples from a subject from which cells can be obtained include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, stool, semen, vaginal fluid, interstitial fluid from tumor tissue, ocular fluid, sweat, mucus, earwax, grease, glandular secretions, spinal fluid, hair, nails, plasma, nasal swab or nasopharyngeal washing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, pharyngeal fluid, sputum, pus, microorganisms, meconium, breast milk, and / or other excrement or body tissue.

[0103] In some cases, the cells may be a population of T cells, NK cells, B cells, etc. obtained from a subject. T cells can be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a blood unit drawn from a subject using any number of techniques, such as Ficoll™ separation. In certain embodiments, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma portion and to place the cells in an appropriate buffer or medium for subsequent processing steps.

[0104] Any of a variety of immune cells may be utilized in the embodiments herein. In some embodiments, immune cells include granulocytes (e.g., basophils, eosinophils, and neutrophils), mast cells, monocytes that can develop into macrophages, antigen-presenting cells (e.g., dendritic cells), and lymphocytes (e.g., natural killer cells (NK cells), B cells, and T cells). In some embodiments, the immune cells are immune effector cells. Immune effector cells refer to immune cells that can perform a specific function in response to a stimulus. In some embodiments, the immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. T cells include naive and memory cells (e.g., central memory or TCM, effector memory or TEM, and effector memory RA or TEMRA), effector cells (e.g., cytotoxic T cells or CTL, or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, THF), regulatory cells (e.g., Treg and Tr1 cells), natural killer T cells (NKT cells), tumor-infiltrating lymphocytes (TIL), activated lymphocyte killer cells (LAK), αβ T cells, γδ T cells, and similar unique types of T cell lineages. T cells are broadly classified into two types: CD8+ T cells and CD4+ T cells, depending on the proteins present on their cell surface. T cells expressing the target system can perform multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTL). CTLs expressing the target system can be involved in the recognition and elimination of virus-infected or cancer cells. CTLs have specialized compartments or granules that contain cytotoxins that trigger apoptosis (e.g., programmed cell death).CD4+ T cells can be divided into four subsets: Th1, Th2, Th17, and Treg, although additional subsets may exist. "Th" refers to "helper T cells." Th1 cells can coordinate immune responses against intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alert and activate other immune cells, such as macrophages, which phagocytose bacteria. Th2 cells are involved in coordinating immune responses against extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin-17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important in neutrophil recruitment.

[0105] In some embodiments, the immune cell populations provided herein may be of heterogeneous origin. In some embodiments, the cells used may consist of a heterogeneous mixture of CD4 and CD8 T cells. The CD4 and CD8 cells may have phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells may also have phenotypic characteristics of effector memory cells. In some embodiments, the cells may be central memory cells.

[0106] In some embodiments, the cells include peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subsets, such as, but not limited to, T cells, natural killer cells, monocytes, naive cells, killer T cells, monocyte precursor cells, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cell, including any T cell, such as tumor-infiltrating cells (TILs), such as CD3+ T cells, CD4+ T cells, CD8+ T cells, or any other type of T cell. T cells can also include memory T cells, stem cell memory T cells, or effector T cells. T cells can be selected from a bulk population, such as by selecting T cells from whole blood. T cells can be expanded from a bulk population. T cells can be biased toward a specific population and phenotype. For example, T cells may be phenotypically biased to include CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+). Suitable cells can be selected that contain one or more markers selected from the list including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+). Cells further include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. Cells can include any number of primary cells (e.g., human cells, non-human cells, and / or mouse cells). Cells can also be progenitor cells. Cells can be from the subject (e.g., patient) to be treated. Cells can be derived from a human donor. The host cells may be stem cell-like memory cells (TSCM) consisting of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, which may express CD95, IL-2Rβ, CXCR3, and LFA-1 and may exhibit many functional attributes distinct from the stem memory cells. The host cells may be central memory cells (TCM) containing L-selectin and CCR7, which may secrete, for example, IL-2 but not IFNγ or IL-4.The cells may also be effector memory cell TEMs that contain L-selectin and CCR7 and produce effector cytokines such as IFNγ and IL-4.

[0107] In various embodiments of the aspects herein, the immune cells comprise lymphocytes. In some embodiments, the lymphocytes are natural killer cells (NK cells). In some embodiments, the lymphocytes are T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In some embodiments, any number of available T cell lines can be used. Immune cells, such as lymphocytes (e.g., cytotoxic lymphocytes), are preferably autologous, although xenogeneic cells can also be used. T cells can be obtained from a unit of blood collected from a subject using any number of techniques, such as Ficoll isolation. Cells from an individual's circulating blood can be obtained by apheresis or leukapheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma portion and place the cells in an appropriate buffer or medium (e.g., phosphate-buffered saline (PBS)) for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, the apheresis sample may be cleared and the cells directly resuspended in medium. The sample may be provided directly by the subject or indirectly through one or more intermediaries, such as a sample collection service or a healthcare provider (e.g., a doctor or nurse). In some embodiments, separation of T cells from peripheral blood leukocytes includes lysing red blood cells and separating monocytes from peripheral blood leukocytes by centrifugation, e.g., using a PERCOL™ gradient.

[0108] Specific subpopulations of T cells, such as CD4+ or CD8+ T cells, can be further isolated using positive or negative selection techniques. For example, negative selection of T cell populations can be achieved using antibody compositions directed against surface markers specific to the cells being negatively selected. One suitable technique involves cell sorting by negative magnetic immunoadhesion, which utilizes a mixture of monoclonal antibodies directed against cell surface markers present on the cells being negatively selected. For example, to isolate CD4+ cells, the monoclonal antibody mixture can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. The negative selection process can be used to produce a largely homogenous population of desired T cells. In some embodiments, the composition contains a mixture of two or more (e.g., 2, 3, 4, 5, or more) different types of T cells.

[0109] In some embodiments, immune cells are members of an enriched cell population. One or more desired cell types can be enriched by any suitable method, including, but not limited to, treating the cell population to trigger proliferation and / or differentiation into desired cell types, preventing the growth of unwanted cell types, killing or lysing unwanted cell types, or purifying the desired cell types (e.g., affinity column purification to retain desired or unwanted cell types based on one or more cell surface markers). In some embodiments, the enriched cell population is enriched for cytotoxic lymphocytes, which are selected from cytotoxic T cells (also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, and killer T cells), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.

[0110] Cell and surface (e.g., particle, such as beads) concentrations can be varied to isolate desired cell populations by positive or negative selection. In some embodiments, it may be necessary to significantly reduce the volume of beads and cells mixed together (i.e., increase cell concentration) to ensure maximum cell-to-bead contact. For example, a concentration of 2 billion cells / mL may be used. In some embodiments, a concentration of 1 billion cells / mL is used. In some embodiments, concentrations greater than 100 million cells / mL are used. Cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL may be used. In other embodiments, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / mL may be used. In further embodiments, concentrations of 1.25 or 1.5 billion cells / mL may be used. The use of higher concentrations results in increased cell yield, cell activation, and cell proliferation.

[0111] The disclosed systems and methods can be used to kill a variety of target cells. Target cells that can be used with the methods include a wide range of cell types. Target cells can be in vitro. Target cells can be in vivo. Target cells can be ex vivo. Target cells can be isolated cells. Target cells can be cells in vivo. Target cells can be cells in cell culture. Target cells can be a collection of cells. Target cells can be mammalian cells or derived from mammalian cells. Target cells can be rodent cells or derived from rodent cells. Target cells can be human cells or derived from human cells. Target cells can be prokaryotic cells or derived from prokaryotic cells. Target cells can be bacterial cells or derived from bacterial cells. Target cells can be archaeal cells or derived from archaeal cells. Target cells can be eukaryotic cells or derived from eukaryotic cells. Target cells can be pluripotent stem cells. Target cells can be plant cells or derived from plant cells. The target cell may be an animal cell or derived from an animal cell. The target cell may be an invertebrate cell or derived from an invertebrate cell. The target cell may be a vertebrate cell or derived from a vertebrate cell. The target cell may be a microbial cell or derived from a microbial cell. The target cell may be a fungal cell or derived from a fungal cell. The target cell may be derived from a specific organ or tissue.

[0112] The target cell may be a stem cell or progenitor cell. The target cell may include a stem cell (e.g., adult stem cell, embryonic stem cell, induced pluripotent stem (iPS) cell) or a progenitor cell (e.g., cardiac progenitor cell, neural progenitor cell, etc.). The target cell may include a mammalian stem cell or progenitor cell, including a rodent stem cell, a rodent progenitor cell, a human stem cell, a human progenitor cell, etc. A clonal cell may include the progeny of a cell. The target cell may include the target nucleic acid. The target cell may be in vivo. The target cell may be a genetically modified cell. The target cell may be a host cell.

[0113] The target cells may be primary cells. For example, the primary cell culture may be at passage 0, 1, 2, 4, 5, 10, 15, or more. The cells may be unicellular organisms. The cells may be grown in culture medium.

[0114] The target cells may be diseased cells. Diseased cells may have altered metabolism, gene expression, and / or morphological characteristics. Diseased cells may be cancer cells, diabetic cells, or apoptotic cells. Diseased cells may be derived from cells of a diseased subject. Exemplary diseases may include blood diseases, cancer, metabolic diseases, eye diseases, organ diseases, musculoskeletal diseases, heart diseases, etc.

[0115] If the target cells are primary culture cells, they can be collected from an individual by any method. For example, white blood cells can be collected by apheresis, leukapheresis, density gradient separation, etc. Cells can be collected by biopsy from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, and stomach. An appropriate solution can be used to separate or suspend the collected cells. Such solutions are typically balanced salt solutions (e.g., saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.) appropriately supplemented with fetal bovine serum or other naturally occurring factors, along with a low concentration of an acceptable buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, etc. Cells can be used immediately or preserved (e.g., by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, media buffer (e.g., 10% DMSO, 50% serum, 40% buffered media), and / or other similar solutions commonly used to preserve cells at freezing temperatures.

[0116] Non-limiting examples of cells that can be target cells include lymphocytes (e.g., B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., U.S. Patent Application Publication No. 20080241194)), myeloid cells (granulocytes (basophils, eosinophils, neutrophils / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells), cells from the endocrine system (thyroid (thyroid epithelial cells, parafollicular cells), parathyroid ( Parathyroid chief cells, Oxyphil cells), adrenal glands (chromaffin cells), pineal gland (pineal cells), nervous system cells (glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Boettcher cells, and pituitary gland (gonadotropins, corticoids, thyroid hormones, growth hormones, lactate hormones)), respiratory system cells (lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells), circulatory system cells (cardiac muscle cells, pericytes), digestive system cells (stomach (gastric chief cells, parietal cells, goblet cells, Enteroendocrine cells (including Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells), enteroendocrine cells (including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle), bone cells (including osteoblasts, osteocytes, osteoclasts, teeth (osteoblasts, ameloblasts)), chondrocytes (including chondrocytes, chondrocytes), skin cells (hair matrix cells, keratinocytes, melanocytes (nevus cells)), muscle cells (including muscle cells), urinary system cells (including podocytes, juxtaglomerular cells, glomerular mesangial cells / glomerular mesangial cells, proximal tubule brush border cells, and macular cells) ), germ line cells (including sperm, Sertoli cells, Leydig cells, and eggs), and other cells (adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, hair matrix cells (stem cells), stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina,Basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina; urothelial cells (bladder and urethra); secretory epithelial cells of the exocrine system; mucous cells of the salivary glands (secreting polysaccharide-rich secretions); serous cells of the salivary glands (secreting glycoprotein enzymes-ri)ch); Von Ebner's gland cells in the tongue (cleansing taste buds); mammary gland cells (secreting milk); lacrimal gland cells (secreting tears); waxy gland cells in the ear (secreting earwax); dark cells of the eccrine sweat glands (secreting glycoproteins); clear cells of the eccrine sweat glands (secreting small molecules). Apocrine sweat gland cells (scented secretions, sensitive to sex hormones), Moll's glands of the eyelids (special sweat glands), sebaceous gland cells (lipid-rich sebum secretions), Bowman's gland cells of the nose (cleansing the olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal fluid components, including fructose for sperm swimming), prostate gland cells (secreting seminal fluid components), Bulbourethral gland cells (mucus secretion), Bartholin's gland cells (secreting vaginal lubrication), Littre's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), goblet cells isolated from the respiratory and digestive tracts (mucus secretion), gastric mucosal cells (mucus secretion), gastric zymogen cells (pepsinogen secretion), gastric acid-secreting cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive fluid secretion). Enzyme-secreting), small intestinal Paneth cells (lysozyme-secreting), lung type II pneumocytes (surfactant-secreting), lung Clara cells, hormone-secreting cells, anterior pituitary cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, pars intermedia cells of the pituitary gland, magnocellular neurosecretory cells, intestinal and respiratory system cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphyllin cells, adrenal cells, chromaffin cells, Ley cells of the testis, ovarian interstitial cells, ovarian follicular rupture lutein cells, granulosa lutein cells, Theca lutein cells, Juxtaglomerular cells (renin-secreting), kidney Macula densa cells, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney, type I pneumocytes (lining the air spaces of the lungs), pancreatic duct cells (central cells), striated duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining sealed body cavities, ciliated cells with propulsive functions,Extracellular matrix-secreting cells, contractile cells, skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (lymphoid tissue), microglial cells (central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, inhibitory T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and unipotent progenitor cells (various types) used in the blood and immune system, pluripotent stem cells, Totipote Examples of target cells include, but are not limited to, nt stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomic nerve cells, sensory and peripheral nerve support cells, central nerve cells and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, ogonium / oocyte, spermatid, spermatocyte, spermatogonium cells (spermatocyte stem cells), sperm, nurse cells, ovarian follicular cells, Sertoli cells (testes), thymic epithelial cells, interstitial cells and kidney stromal cells, or engineered or irradiated non-human cells such as K562 and NK92. Target cells may be natural or modified cells.

[0117] Of particular interest are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastoma, anal carcinoma, and anaplastic large cell lymphoma. , anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, vascular leiomyolipomas, angiosarcomas, adnexal carcinoma, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal cell-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, malignant bone tumor, benign bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown tumor, Burkitt lymphoma, carcinoma of unknown primary origin, carcinoid tumor, cancer, carcinoma in situ, penile cancer, carcinoma of unknown primary origin, Cancer sarcoma, Castleman's disease, central nervous system embryonal tumors, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic granulocytic leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell sarcoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma, dermoid cyst, desmoplastic small cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor tumor, endometrial cancer, endometrial carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymomas, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing tumor family, Ewing sarcoma family, Ewing sarcoma, extracranial germ cell tumor, extrahepatic germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal tumor, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, glioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer,Gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, embryonal tumor, gestational choriocarcinoma, gestational trophodermal tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral glioma, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, malignant cardiac tumor, hemangioblastoma, hemangiopericytoma, angiosarcoma, malignant cardiac tumor, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer , Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myeloid leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteinizing cell tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma , malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma, occult primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, myxoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloid leukemia, myeloma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, tumor, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, schwannoma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligolymphoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer or al cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low malignant potential tumor of the ovary, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papilloma, paraganglioma,Paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineocytoma, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, polyblastoma, T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal Cancer, renal cell carcinoma, malignant tumors of the respiratory tract related to the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannoma, sebaceous gland carcinoma, secondary tumors, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, Intestinal cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial and stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal malignant lymphoma, testicular cancer, Thec Cancer cells include sarcoma, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary system tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subgroup within a cancer cell population, e.g., cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, e.g., lymphoma. The antigen may be a tumor-associated antigen.

[0118] In some embodiments, the target cells form tumors. Tumors treated using the methods herein can result in stable tumor growth (e.g., one or more tumors increase in volume by less than 1%, 5%, 10%, 15%, or 20%, and / or do not metastasize). In some embodiments, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. In some embodiments, the tumor size or tumor cell number is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated or reduced to below detectable levels. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after treatment.

[0119] Determining target cell death can be done by any suitable method, including, but not limited to, counting cells before or after treatment, or measuring the level of a marker for live or dead cells (e.g., live or dead target cells). Measuring cell death can be done by any suitable method. In some embodiments, cell death is measured relative to initial conditions. For example, an individual may have a known initial amount of target cells (e.g., an initial cell population of known size or a known concentration of circulating target cells). In such cases, cell death can be expressed as the ratio of surviving cells to the initial cell population after treatment. In some embodiments, cell death can be measured by a suitable cell death assay. A variety of cell death assays are available, and various detection methods can also be utilized. Examples of detection methods include, but are not limited to, cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof.

[0120] After the treatment period, if the tumor is surgically resected, the therapeutic effect in terms of tumor size reduction can be determined by measuring the percentage of resected tissue that is necrotic (i.e., dead). In some embodiments, a therapeutically effective treatment is one in which the necrotic rate of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the necrotic rate of the resected tissue is 100%, i.e., there is no viable tumor tissue or no detectable viable tumor tissue.

[0121] Exposure of target cells to the immune cells or immune cells disclosed herein can be carried out in vitro or in vivo. Exposure of target cells to immune cells or immune cells generally refers to contacting the target cells with the immune cells and / or bringing them into sufficient proximity to allow antigens (e.g., membrane-bound or non-membrane-bound) of the target cells to bind to enhanced receptors expressed on the immune cells. Exposure of target cells to immune cells or immune cells also refers to contacting the target cells with the immune cells and / or bringing them into sufficient proximity to allow antigens (e.g., membrane-bound or non-membrane-bound) of the target cells to bind to CARs expressed in the immune cells. Exposure of target cells to immune cells or immune cells in vitro to allow binding to CARs expressed in the immune cells can be achieved by co-culturing the target cells and immune cells. The target cells and immune cells can be co-cultured, for example, as adherent cells or alternatively in suspension. The target cells and immune cells can be co-cultured in various suitable types of cell culture media supplemented, for example, with supplements, growth factors, ions, etc. Exposing target cells to an immune cell or cells in vivo can, in some cases, be accomplished by administering the immune cells to a subject (e.g., a human subject) and allowing the immune cells to localize to the target cells via the circulatory system. In some cases, the immune cells can be delivered, for example, by direct injection, to the direct area where the target cells are localized.

[0122] The exposure can be for any suitable period of time, such as at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or more.

[0123] The various domains of the enhanced receptors and CARs provided herein can be linked by chemical bonds, such as amide or disulfide bonds, small organic molecules (e.g., hydrocarbon chains), amino acid sequences, such as peptide linkers (e.g., amino acid sequences about 3 to 200 amino acids in length), or a combination of small organic molecules and peptide linkers. The peptide linker can provide the desired flexibility to allow for the desired expression, activity, and / or conformational position of the chimeric polypeptide. The peptide linker can be of any appropriate length to connect at least two target domains and is preferably designed to be sufficiently flexible to allow for the correct folding and / or function and / or activity of the one or two domains it connects. The peptide linker can be at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. In some embodiments, the length of the peptide linker is about 0-200 amino acids, about 10-190 amino acids, about 20-180 amino acids, about 30-170 amino acids, about 40-160 amino acids, about 50-150 amino acids, about 60-140 amino acids, about 70-130 amino acids, about 80-120 amino acids, or about 90-110 amino acids. In some embodiments, the linker sequence may comprise a sequence of an endogenous protein. In some embodiments, the linker sequence comprises glycine, alanine, or serine amino acid residues. In some embodiments, the linker may comprise a GS, GGS, GGGGS, GGSG, or SGGG motif, e.g., multiple repeats of the motif. The linker sequence may comprise any naturally occurring amino acid, non-naturally occurring amino acid, or a combination thereof.

[0124] Any suitable delivery method can be used to introduce the disclosed compositions and molecules (e.g., polypeptides and / or nucleic acids encoding polypeptides) into host cells, such as immune cells. The various components can be delivered simultaneously or temporally separately. The choice of method can depend on the type of cell to be transformed and / or the environment in which the transformation is performed (e.g., in vitro, ex vivo, or in vivo).

[0125] Delivery methods can include contacting a target polynucleotide or introducing a nucleic acid comprising a nucleotide sequence encoding one or more compositions of the present disclosure into a cell (or a group of cells, such as an immune cell). Suitable nucleic acids comprising a nucleotide sequence encoding a composition of the present disclosure can comprise an expression vector, where an expression vector comprising a nucleotide sequence encoding one or more compositions of the present disclosure is a recombinant expression vector.

[0126] Non-limiting examples of delivery methods or transformation include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0127] In some embodiments, the present disclosure provides methods that include delivering one or more polynucleotides, or one or more vectors described herein, or one or more transcription products thereof, and / or one or more proteins transcribed therefrom, to a host cell. In some embodiments, the present disclosure further provides cells produced by these methods, and organisms (e.g., animals, plants, or fungi) that comprise or are produced by these cells.

[0128] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. These methods can be used to administer nucleic acids encoding the compositions of the present disclosure to cells in culture or host organisms. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., transcription products of the vectors described herein), naked nucleic acids, and nucleic acids incorporated into delivery vectors such as liposomes. Viral vector delivery systems can include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells.

[0129] Non-viral methods for nucleic acid delivery can include lipofection, nucleofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, and drug-enhanced uptake of DNA. Cationic and neutral lipids suitable for effective receptor-recognition lipofection of polynucleotides can be used. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). Lipids can be used to prepare lipid:nucleic acid complexes, including targeted liposomes, such as immunolipid complexes.

[0130] RNA or DNA virus-based systems can be used to target specific cells in the body and deliver viral payloads to the cell nucleus. Viral vectors can be administered directly (in vivo) or used to treat cells in vitro, and optionally, modified cells can be administered (ex vivo). Viral-based systems can include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors used for gene transfer. Retroviral, lentiviral, and adeno-associated virus gene transfer methods can be used to integrate into the host genome, resulting in long-term expression of the inserted transgene. High transduction efficiencies can be observed in many different cell types and target tissues.

[0131] Lentiviruses can integrate their genomes into host cells (e.g., 293 cells or T cells). Lentiviruses can use a three-plasmid system or a four-plasmid system.

[0132] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and produce high viral titers. The choice of retroviral gene delivery system can depend on the target tissue. Retroviral vectors can contain cis-acting long terminal repeats capable of packaging foreign sequences up to 6-10 kb. Minimal cis-acting LTRs can be sufficient for vector replication and packaging, which can be used to integrate therapeutic genes into target cells and provide permanent transgene expression. Retroviral vectors can include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0133] Adenovirus-based systems can be used. Adenovirus-based systems can result in transient expression of transgenes. Adenovirus-based vectors can have high transduction efficiency in cells and do not require cell division. High titers and expression levels can be obtained using adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce cells with target nucleic acids, for example, for in vitro nucleic acid and peptide production, and can also be used in in vivo and ex vivo gene therapy processes.

[0134] Packaging cells can be used to form viral particles capable of infecting host cells. Such cells can include 293 cells (e.g., for lentivirus or adenovirus packaging) and Psi2 or PA317 cells (e.g., for retrovirus packaging). Viral vectors can be produced by generating cell lines that package nucleic acid vectors into viral particles. The vectors can contain the minimal viral sequences required for packaging and subsequent integration into the host. The vectors can contain other viral sequences substituted with an expression cassette for the polynucleotide to be expressed. Missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors can contain ITR sequences from the AAV genome, which are essential for packaging and integration into the host genome. Viral DNA can be packaged in cell lines that lack the ITR sequences but contain helper plasmids encoding other AAV genes, namely rep and cap. Cell lines can also be infected with adenovirus as helper cells. The helper virus can facilitate AAV vector replication and expression of AAV genes from the helper plasmid. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Other methods for delivering nucleic acids to cells can also be used, for example, as described in U.S. Patent Application Publication No. 20030087817, which is incorporated herein by reference.

[0135] One or more vectors described herein can be used to transiently or non-transiently transfect host cells. Cells can be transfected as they naturally occur in a subject. Cells can be transfected as they are obtained from or derived from a subject. Cells can be derived from cells obtained from a subject, such as cell lines. In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines containing sequences from one or more vectors. In some embodiments, cells transiently transfected with a composition of the present disclosure (e.g., by transient transfection of one or more vectors or by RNA transfection) can be used to establish new cell lines, including cells containing modifications but lacking any other exogenous sequences.

[0136] Any suitable vector compatible with the host cell can be used with the methods of the present disclosure. Non-limiting examples of vectors for eukaryotic host cells include pXT1, pSG5 (Stratagene™), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia™).

[0137] Contact of cells with the composition can occur in any medium and under any culture conditions that promote cell survival. For example, cells can be suspended in any convenient and suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640 supplemented with fetal bovine serum or heat-inactivated goat serum (approximately 5-10%), L-glutamine, thiols (particularly 2-mercaptoethanol), and antibiotics (e.g., penicillin and streptomycin). The culture can also contain growth factors to which the cells are responsive. As defined herein, growth factors are molecules that can promote cell survival, growth, and / or differentiation in culture or in intact tissues by specific action on transmembrane receptors. Growth factors can include polypeptide and non-polypeptide factors.

[0138] In many embodiments, the selected delivery system targets specific tissue or cell type.In some cases, the delivery system is bound to tissue or cell specific marker (for example, cell surface protein) to achieve tissue or cell targeting.Viral and non-viral delivery systems can be customized to target tissue or cell type of interest.

[0139] Pharmaceutical compositions containing the molecules (e.g., polypeptides and / or nucleic acids or proteins encoding the polypeptides) or immune cells described herein can be administered for prophylactic and / or therapeutic therapy. In therapeutic use, the compositions can be administered to a subject already suffering from a disease or disorder in an amount sufficient to cure or at least partially arrest, or to cure, heal, ameliorate, or improve the condition of, the symptoms of the disease or disorder. Effective amounts for such use can vary based on the severity and course of the disease or disorder, previous treatments, the subject's health, weight, and response to drugs, and the judgment of the treating physician.

[0140] The therapeutic agents may be administered in any order or simultaneously. If administered simultaneously, the therapeutic agents may be provided in a single combined form or in multiple forms (e.g., multiple individual tablets or cell solutions). The molecules and cell solutions may be packaged together or separately, in a single package or multiple packages. One or all of the therapeutic agents may be administered multiple times. If not administered simultaneously, the interval between multiple doses may vary from approximately 1 to 24 months.

[0141] The molecules or cells described herein can be administered before, during, or after the onset of a disease or disorder, and the timing of administration of compositions containing the compounds can vary. For example, pharmaceutical compositions can be used as prophylactics and can be administered continuously to subjects prone to a disorder or disease to prevent the onset of the disease or disorder. The molecules, cells, and pharmaceutical compositions can be administered to a subject during the onset of symptoms or as soon as possible. Administration of the molecules can begin within the first 48 hours of the onset of symptoms, within 24 hours of the onset of symptoms, within 6 hours of the onset of symptoms, or within 3 hours of the onset of symptoms. Symptoms occur. Initial administration can be by any practical route. For example, any of the formulations described herein can be used by any of the routes described herein. The molecules can be administered as soon as possible after the onset of a disease or disorder is detected or suspected, and for the period necessary to treat the disease (e.g., about 1 month to about 3 months). The duration of treatment can vary for each subject.

[0142] The molecule can be packaged in a biological compartment, and the biological compartment containing the molecule can be administered to a subject. Biological compartments include, but are not limited to, viruses (lentivirus, adenovirus), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles.

[0143] For example, the biological compartment may include a liposome. A liposome may be a self-assembled structure comprising one or more lipid bilayers, each of which may comprise two monolayers of amphiphilic lipid molecules oriented in opposite directions. The amphiphilic lipid may comprise a polar (hydrophilic) head group covalently attached to one or more nonpolar (hydrophobic) acyl or alkyl chains. Energetically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium may induce self-organization of the amphiphilic lipid molecules, orienting the polar head groups toward the surface of the bilayer and the acyl chains toward the interior of the bilayer, effectively shielding the acyl chains from contact with the aqueous environment.

[0144] Glycerophospholipids and sphingolipids used in liposomes may include, representative examples thereof include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phoasphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoyl lecithin; preferred examples of amphiphilic compounds include DPPC, dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, and egg sphingomyelin, or any combination thereof.

[0145] The biological compartment can include nanoparticles, which can have a diameter of about 40 nanometers to about 1.5 micrometers, about 50 nanometers to about 1.2 micrometers, about 60 nanometers to about 1 micrometer, about 70 nanometers to about 800 nanometers, about 80 nanometers to about 600 nanometers, about 90 nanometers to about 400 nanometers, or about 100 nanometers to about 200 nanometers.

[0146] In some cases, increasing the size of the nanoparticles can slow or prolong the release rate, and decreasing the size of the nanoparticles can increase the release rate.

[0147] The amount of albumin in the nanoparticles can be about 5% to about 85% (v / v) albumin, about 10% to about 80%, about 15% to about 80%, about 20% to about 20%, about 70% albumin (v / v), about 25% to about 60%, about 30% to about 50%, or about 35% to about 40%. The pharmaceutical composition can contain up to 30, 40, 50, 60, 70, or 80% or more nanoparticles. In some cases, the nucleic acid molecules of the present disclosure can be bound to the surface of the nanoparticles.

[0148] The biological compartment can include a virus. The virus can be a delivery system for the pharmaceutical composition of the present disclosure. Exemplary viruses include lentivirus, retrovirus, adenovirus, herpes simplex virus I or II, parvovirus, reticuloendotheliosis virus, and adeno-associated virus (AAV). The pharmaceutical composition of the present disclosure can be delivered to cells using a virus. The virus can infect and transduce cells in vivo, ex vivo, or in vitro. In ex vivo and in vitro transduction, the transduced cells can be administered to a subject in need of treatment.

[0149] The pharmaceutical composition can be packaged in a viral delivery system, for example, the composition can be packaged into a viral particle using an HSV-1 helper virus-free packaging system.

[0150] The viral delivery system (e.g., a virus comprising a pharmaceutical composition of the present disclosure) can be administered to cells, tissues, or organs of a subject in need thereof by direct intraventricular injection, stereotactic injection, by micropump infusion system, convection, catheter, intravenous, parenteral, intraperitoneal, and / or subcutaneous injection. In some cases, cells can be transduced in vitro or ex vivo using the viral delivery system. The transduced cells can be administered to a subject with a disease. For example, stem cells can be transduced with the viral delivery system comprising the pharmaceutical composition, and the stem cells can be transplanted into a patient's body to treat the disease.

[0151] In some cases, the dose of cells administered to the subject is 1×10 4 cells / kg or less, or approximately 1 × 10 4 cells / kg, approximately 2 × 10 4 cells / kg, approximately 3 × 10 4 cells / kg, approximately 4 × 10 4 cells / kg, approximately 5 × 10 4 cells / kg, approximately 6 × 10 4 cells / kg, approximately 7 × 10 4 cells / kg, approximately 8 × 10 4 cells / kg, approximately 9 × 10 4 cells, approximately 1 × 10 5 cells / kg, approximately 2 × 10 5 cells / kg, approximately 3 × 10 5 cells / kg, approximately 4 × 10 5 cells / kg, approximately 5 × 10 5 cells / kg, approximately 6 × 10 5 cells / kg, approximately 7 × 10 5 cells / kg, approximately 8 × 10 5 cells / kg, approximately 9 × 10 5 cells, approximately 1 × 10 6 cells / kg, approximately 2 × 10 6 cells / kg, approximately 3 × 10 6 cells / kg, approximately 4 × 10 6cells / kg, approximately 5 × 10 6 cells / kg, approximately 6 × 10 6 cells / kg, approximately 7 × 10 6 cells / kg, approximately 8 × 10 6 cells / kg, approximately 9 × 10 6 cells, approximately 1 × 10 7 cells / kg, approximately 5 × 10 7 cells / kg, approximately 1 × 10 8 The cell dosage may be calculated based on the number of successfully transfected, effectively modified cells, or may be calculated based on the total number of cells.

[0152] Biological compartments can be introduced into cells by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0153] In some embodiments, immune cells expressing a subject system are administered. The immune cells expressing a subject system can be administered before, during, or after the onset of a disease or disorder, and the timing of immune cell administration can vary. For example, immune cells expressing a subject system can be used as a prophylactic and can be administered continuously to a subject with a disorder or predisposition to a disease to prevent the onset of the disease or disorder. The immune cells can be administered to a subject during the onset of symptoms or as soon as possible. Administration can begin within the first 48 hours of symptom onset, within the first 24 hours of symptom onset, within the first 6 hours of symptom onset, or within 3 hours of symptom onset. Initial administration can be by any appropriate route, for example, by any route described herein, using any formulation described herein. The immune cells can be administered as soon as possible after the onset of a disease or disorder is detected or suspected, and for a period of time necessary to treat the disease (e.g., about one month to about three months). The duration of treatment can vary for each subject.

[0154] The molecules (e.g., polypeptides and / or nucleic acids) described herein may be present in an amount of about 1 mg to about 2000 mg, about 5 mg to about 1000 mg, about 10 mg to about 25 mg to 500 mg, about 50 mg to about 250 mg, about 100 mg to about 200 mg, about 1 mg to about 50 mg, about 1 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, or about 300 mg to about 350 mg. mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, about 450 mg to about 500 mg, about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg to about 950 mg, or about 950 mg to about 1000 mg.

[0155] The molecules (e.g., polypeptides and / or nucleic acids) described herein may be administered in amounts of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 ... mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.

[0156] The molecules (e.g., polypeptides and / or nucleic acids) described herein can be present in a composition that provides at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 10 or more activity / mg molecule units. The activity can be modulation of gene expression. In some embodiments, the total active units of the molecules delivered to the subject is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units. In some embodiments, the total number of active units of the molecule delivered to the subject is at most 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units. [Brief explanation of the drawings]

[0157] [Figure 1] FIG. 1 shows an in vitro experiment comparing the enhancement of T cell function (IFNγ secretion) by various potentiating receptors. [Figure 2] FIG. 1 shows an in vitro experiment comparing the improvement of T cell function (secretion of IL2) by various potentiating receptors. [Figure 3] FIG. 1 shows animal experiments comparing the enhancement of tumor suppressor function by T cells with various enhanced receptors. DETAILED DESCRIPTION OF THE INVENTION

[0158] For a full understanding and utilization of the present invention, the present invention will be described in detail in the following with reference to examples and the accompanying drawings. The examples are intended to illustrate and explain the present invention only and are not intended to limit the scope of the present invention, which is specifically limited by the appended claims.

[0159] Example 1. Example regarding the enhanced receptor - PD1 / CD28 The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of PD1 and an intracellular domain of CD28, and is referred to as PD1 / CD28 or ER (Enhanced Receptor).

[0160] Preparation of lentiviral vectors of PD1 / CD28-enhanced receptor The PD1 / CD28-enhanced receptor (hereafter referred to as "Switch") lentiviral vector was developed using a safer, fourth-generation lentiviral vector system. The main vector PD1 / CD28, the packaging vectors pMDL-gag and Rev, and the envelope vector pMD2.G were co-transfected into 293T cells using calcium phosphate or liposomal PEI. After 48 hours, the supernatant was collected and the lentivirus was concentrated by ultracentrifugation.

[0161] The titer of the PD1 / CD28-enhanced receptor lentiviral vector was measured by 3-fold dilution, and 50 μl of the vector was taken and used to infect 293T cells for 48 to 72 hours. The 293T cells were then harvested and stained for PD1. The proportion of PD1+ cells was analyzed by flow cytometry, and the titer was calculated based on the formula.

[0162] Titer (TU / ml) = 40,000-45,000 (initial 293T cell count) x PD1 + Cell percentage × dilution factor × 20 (first PD1 <20%) + Calculated using cell percentage).

[0163] Similarly, a lentiviral vector containing the following gene sequence was constructed:

[0164] 1) Non-fused expression of an artificial TCR targeting PD1 / CD28 and the tumor target NY-ESO-1, termed ER-TCR.

[0165] 2) Non-fusion expression of CAR targeting PD1 / CD28 and CD19, termed ER-CAR.

[0166] Calculate the titer of each lentivirus and incubate until the titer is >3 x 10 7 If so, it can be used in the next step.

[0167] Construction of other groups of lentiviral expression vectors Similar to the above steps, lentiviral vectors carrying the following gene sequences were constructed.

[0168] 1) An artificial TCR targeting the tumor target NY-ESO-1.

[0169] 2) CARs targeting CD19.

[0170] 4) Non-fused expression of an artificial TCR targeting PD1 / CD28 and the tumor target NY-ESO-1, termed ER-TCR.

[0171] 5) Non-fusion expression of CAR targeting PD1 / CD28 and CD19, termed ER-CAR.

[0172] Preparation of several populations of tumor-distinguishing T cells Four types of tumor-discriminating T cells were prepared as follows:

[0173] Preparation of TCR-T cells (NY-ESO-1-TCR-T or TCR-T) targeting tumor target NY-ESO-1 Patient leukocytes containing NY-ESO-1-positive, HLA-A:0201-positive tumor cells were collected by apheresis. PBMCs were isolated using Ficoll lymphocyte separation medium and grown for 2 hours. Suspended cells, primarily T cells, were then collected. A TCR lentiviral vector recognizing NY-ESO-1 was added at an MOI (number of lentiviruses / number of cells) of 1, and TCR-T cells were cultured and expanded. Flow cytometry revealed that 68.7% of patients expressed the TCR.

[0174] ER lentivirus was added to TCR-T at an MOI of 1 to obtain ER-TCR-T.

[0175] Preparation of CD19-targeting CAR-T cells (CD19CAR-T or CAR-T) and enhanced receptor-transfected CAR-T (ER-CAR-T) Patients' white blood cells were collected by apheresis, and PBMCs were isolated using Ficoll lymphocyte separation medium. After two hours of adherent growth, floating cells (mainly T cells) were collected. A CD19-recognizing CAR lentiviral vector was added at an MOI (number of lentiviruses / number of cells) of 1, and CAR-T cells were cultured and expanded.

[0176] ER lentivirus was added to CAR-T at an MOI of 1 to obtain ER-CAR-T.

[0177] Preparation of fresh tumor tissue-derived TIL cells and enhanced receptor-transfected TILs (ER-TILs) Tumor tissue cells were isolated from 10 grams or more of the patient's tumor tissue by enzymatic digestion, CD3-positive T cells (TILs) were isolated and purified using CD3 magnetic beads, and the remaining cells were cultured in adherence to obtain the patient's autologous tumor cells.

[0178] ER lentivirus was added to TILs at an MOI of 1 to obtain ER-TILs.

[0179] Preparation of neoantigen-reactive T cells and enhanced receptor-transfected neoT (ER-neoT) First, whole-exome or RNA transcriptome sequencing was performed on the patient's peripheral blood or surgically resected tumor tissue. Mutation sites were selected, and affinity peptide prediction was performed in combination with the patient's HLA type. Approximately 20 mutations were selected as neoantigens. The neoantigens were gene-synthesized and transcribed into RNA in vitro. Again, the patient's peripheral blood was collected by apheresis or directly, and PBMCs were isolated and adherently expanded for 2 hours. To promote DC differentiation and maturation, adherent monocytes were supplemented with relevant cytokines, and RNA was introduced into the cells by electroporation. Suspension cells, primarily T cells, were co-cultured with electroporated transformed DCs. CD137+ T cells were isolated and expanded to obtain neoantigen-reactive T cells (neoT).

[0180] ER lentivirus was added to neoT at an MOI of 1 to obtain ER-neoT.

[0181] In vitro functional testing of PD1 / CD28-enhanced receptors in TCR-T targeting NY-ESO-1 To verify the role of PD1 / CD28-reinforced receptors in TCR-T targeting NY-ESO-1, we first constructed a tumor cell line, J82-NY-ESO-1-PDL1, with HLA type A:0201. Lentivirus carrying PDL1 and NY-ESO-1 was added to J82 cells at an MOI of 5. At 72 h, positive cells were selected by adding G418 and puromycin. Approximately two weeks later, PDL1 and NY-ESO-1 expression was detected by flow cytometry. Flow cytometry results showed that up to 95% of J82 cells co-expressed PDL1 and NY-ESO-1, indicating successful cell construction.

[0182] To verify the function of ER, we co-cultured tumor cells with T cells to detect CD107a expression and cytokine secretion in T cells. As shown in Figure 6a, CD107a expression in ER-TCR-T cells was higher than that in normal TCR-T cells. IFN-γ and IL-2 secretion also exceeded that of TCR-T cells. In vitro experiments showed that the tumor-killing ability of TCR-T cells was significantly improved after transfection with ER.

[0183] In vitro functional testing of TILs expressing PD1 / CD28-reinforced receptors To verify the effect of Switch on TILs, the inventors co-cultured tumor cells isolated from fresh tumor tissues of patients with TILs and cells for 24 hours, respectively, and detected the secretion of IFN-γ and IL-2 by ELISA. The results showed that the killing ability of ER-TILs was significantly higher than that of TILs not transfected with the enhanced receptor. In vitro experiments showed that the tumor-killing ability of TILs was significantly improved after transfection with ER.

[0184] In vitro functional studies of neoantigen-reactive T cells expressing PD1 / CD28-reinforced receptors To verify the effect of Switch on neoT, the inventors co-cultured tumor cells isolated from fresh tumor tissues of patients with neoT and Switch-neoT cells for 24 hours, respectively, and detected the secretion of IFN-γ and IL-2 by ELISA. The results showed that the killing ability of ER-neoT was significantly higher than that of neoT cells not transfected with the enhanced receptor. In vitro experiments showed that the tumor-killing ability of neoT was significantly improved after transfection with ER.

[0185] In vitro functional studies of CAR-T cells expressing PD1 / CD28-reinforced receptors First, a CD19-expressing tumor cell line was constructed and co-cultured with conventional CD19 CAR-T cells (not transfected with the enhanced receptor) and ER-CAR-T for 24 hours. IFN-γ and IL-2 secretion was detected by ELISA. The killing ability of ER-CAR-T was significantly higher than that of conventional CAR-T (not transfected with the enhanced receptor).

[0186] Animal experiments of NY-ESO-1 TCR-T cells expressing PD1 / CD28-enhanced receptors Inoculation: 1×10 6 The tumor cells J82-NY-ESO-1-PDL1 were subcutaneously inoculated into NSG mice, and a blank control group received a subcutaneous injection of PBS (A0). Tumor formation began in the tumor cell-injected mice approximately two weeks later. Tumor size was measured on day 23, and 30 mice were recruited.

[0187] Administration: PBS was injected into the tail vein of the blank control group (A0), and the mice inoculated with tumor cells were divided into five groups: non-administered PBS group (A1), T cell group (A2), ER-T cell group (A3), TCR-T cell group (A4), and ER-TCR-T cell group (A5). The administration method was 1 × 10 7 The cells were injected intravenously into the tail vein.

[0188] After administration, the tumor size was measured and the condition of the mice was observed every 2 to 3 days, and tumor volume = 1 / 2 x major axis x minor axis x minor axis was observed continuously for 30 days.

[0189] Changes in tumor burden: Tumor volume in tumor-bearing mice continued to increase for one week after administration, and tumor shrinkage was detected in groups A4 and A5 on days 7 to 12. Group A4 began to rebound on days 15 to 25, while group A5 continued to shrink until it nearly disappeared. In the other three groups, tumors maintained their natural growth.

[0190] Changes in total body mass of infused T cells: Of the three groups administered the drug, one mouse was randomly selected from groups 3, 4, and 5 on the 10th day after administration, and tumor tissue was collected to isolate and count T cells (i.e., TILs). The TIL density (i.e., the total number of TILs in the tumor tissue per unit weight) was calculated, and the results showed that the TIL density of the tumors in group A5 was far higher than that of the other groups.

[0191] Animal experiments with CAR-T cells expressing PD1 / CD28-enhanced receptors Inoculation: 1 x 10 CD19-expressing 6 The tumor cells were subcutaneously inoculated into NSG mice, and a blank control group received a subcutaneous injection of PBS (A0). Tumor formation began in the tumor cell-injected mice approximately two weeks later. Tumor size was measured on day 23, and 30 mice were recruited.

[0192] Administration: PBS was injected into the tail vein of the blank control group (A0), and the mice inoculated with tumor cells were divided into five groups: non-administered PBS group (A1), T cell group (A2), ER-T cell group (A3), CAR-T cell group (A4), and ER-CAR-T cell group (A5). All administration methods were 1 × 10 7 The cells were injected intravenously into the tail vein.

[0193] After administration, the tumor size was measured and the condition of the mice was observed every 2 to 3 days, and tumor volume = 1 / 2 x major axis x minor axis x minor axis was observed continuously for 30 days.

[0194] Changes in tumor burden: The tumor volume of the tumor-bearing mice continued to increase in each group for one week after administration. The trend was basically the same in groups A1 to A3, with deaths occurring between the 30th and 40th day. In groups A4 and A5, tumor shrinkage was detected starting from the second week. In group A4, a rebound began between the 3rd and 4th week, but in group A5, tumor shrinkage continued until it almost disappeared.

[0195] Changes in total body mass of infused T cells: Of the three groups administered the drug, one mouse was randomly selected from groups 3, 4, and 5 on the 10th day after administration, and tumor tissue was collected to isolate and count T cells (i.e., TILs). The TIL density (i.e., the total number of TILs in the tumor tissue per unit weight) was calculated, and the results showed that the TIL density of the tumors in group A5 was far higher than that of the other groups.

[0196] Example 2. Example regarding the potentiated receptor - PD1 / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of PD1 and an intracellular domain of 41BBz. This receptor is referred to as PD1 / 41BBz or ER (Enhanced Receptor). The enhanced receptor in this example differs from the enhanced receptor in Example 1 in the costimulatory molecule in the intracellular domain.

[0197] The experimental design is the same as in Example 1, except that the potentiating receptor is different.

[0198] In vitro functional testing of NY-ESO-1-targeting TCR-T expressing PD1 / 41BBz-enhanced receptors The experimental results showed that the expression of CD107a and the secretion of IFN-γ and IL-2 by ER-TCR-T cells were higher than those of normal TCR-T cells that had not been transfected with the enhanced receptor.

[0199] In vitro functional testing of TILs expressing PD1 / 41BBz-enhanced receptors The results of in vitro experiments showed that the ability of TILs to kill tumors was significantly improved after transfection with ER.

[0200] In vitro functional studies of neoantigen-reactive T cells expressing PD1 / 41BBz-enhanced receptors The results showed that the killing ability of ER-neoT was significantly higher than that of neoT.

[0201] In vitro functional studies of CAR-T cells expressing PD1 / 41BBz-reinforced receptors The results showed that the killing ability of ER-CAR-T was significantly higher than that of regular CAR-T that was not transfected with the enhanced receptor.

[0202] Animal experiments of NY-ESO-1 TCR-T cells expressing PD1 / 41BBz-enhanced receptors The experimental design was exactly the same as that of the TCR-T animal experiment in Example 1, with the only difference being the reinforced receptor used. The conclusions of the experiment were similar to those of the TCR-T animal experiment in Example 1: the ER-TCR-T group had more thorough tumor removal than the TCR-T group not transfected with the reinforced receptor (indicating the activity-enhancing properties of ER), the TIL density in the tumor tissue was higher than that of the other groups (indicating that ER can help better concentrate TCR-T cells into tumors), and furthermore, the T cell (ER-T) group transfected with the reinforced receptor but not recognizing tumors failed to kill tumors (indicating the safety of ER; i.e., ER will not independently kill PDL1-positive target cells unless T cells recognize target cells).

[0203] Animal experiments with CD19 CAR-T cells expressing PD1 / 41BBz-enhanced receptors The experimental design was exactly the same as that of the CAR-T animal experiment in Example 1, with the only difference being the reinforced receptor used. The conclusions of the experiment were also similar to those of the CAR-T animal experiment in Example 1: the ER-CAR-T group had more thorough tumor removal than the CAR-T group not transfected with the reinforced receptor (indicating the activity-enhancing properties of ER), the TIL density in the tumor tissue was higher than that of the other groups (indicating that ER can help CAR-T cells better concentrate in tumors), and the reinforced receptor-transfected T cell (ER-T) group, which does not recognize tumors, was unable to kill tumors (indicating the safety of ER; that is, if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0204] Example 3. Example regarding the potentiating receptor - PD1 / ICOS The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of PD1 and an intracellular domain of ICOS, and is referred to as PD1 / ICOS or ER (Enhanced Receptor). The enhanced receptor in this example differs from the enhanced receptor in Example 1 in the costimulatory molecule in the intracellular domain.

[0205] The experimental design is the same as in Example 1, except that the potentiating receptor is different.

[0206] The conclusions of the in vitro experiments were similar to those in Example 1, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0207] The conclusions of the animal experiments were similar to those in Example 1: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that did not recognize tumors transfected with the enhanced receptor (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0208] Example 4. Enhanced Receptor - Anti-HER2 Monoclonal Antibody / CD28 Example In this example, the inventors constructed and tested an enhanced receptor whose extracellular domain is a HER2 anti-monoclonal antibody (abbreviated as aHER2mAb) and whose intracellular domain is CD28. The Fab region of a HER2 antibody is typically designated K4D5. This first type of enhanced receptor is designated aHER2mAb / CD28 or K4D5 / CD28.

[0209] Amino acid sequences of three anti-HER2 antibodies, K4D5, with different affinities K4D5-6.28, K4D5-7.28, and K4D5-8.28 are the Fab regions of three antibodies that specifically recognize HER2, arranged in order of decreasing affinity. Of these, K4D5-7.28 and K4D5-6.28 differ only at amino acid residue 76, while K4D5-8.28 and K4D5-6.28 differ only at amino acid residue 250.

[0210] Construction of three K4D5 / CD28 and K4D5 / CD28-CD19 CAR lentiviral expression plasmids Three K4D5 / CD28 and K4D5 / CD28-CD19 CAR lentiviral expression plasmids were constructed. K4D5 / CD28 and CD19 CARs were expressed as non-fusion constructs.

[0211] Preparation of K4D5 / CD28 and K4D5 / CD28-CD19 CAR lentiviral vectors The three K4D5 / CD28 and K4D5 / CD28-CD19 CAR lentiviral vectors were developed using the safer fourth-generation lentiviral vector system. The main vector K4D5 / CD28 or K4D5 / CD28-CD19 CAR, packaging vectors pMDL-gag and Rev, and envelope vector pMD2.G were co-transfected into 293T cells using lipofectamin 2000. After 48 hours, the supernatants were collected and the lentivirus was concentrated by ultracentrifugation.

[0212] The titer of the three K4D5 / CD28 and K4D5 / CD28-CD19 CAR lentiviral vectors was measured by 3-fold dilution, and 50 μl was taken and used to infect 293T cells for 48 to 72 hours. The 293T cells were then harvested and labeled with HER2 recombinant protein. The proportion of HER2-positive cells was analyzed by flow cytometry, and the titer was calculated based on the formula.

[0213] Titer (TU / ml) = initial 293T cell count × HER2 + Cell percentage × dilution factor × 20 (first HER2 <20%) +(Calculated using the cell percentage.) Using K4D5-8.28 / CD28 as an example, calculate the titer of each lentivirus and determine if the titer is >1 × 10 7 If so, it can be used in the next step.

[0214] Preparation of K4D5 / CD28 enhanced receptor-transfected CAR-T cells Thirty milliliters of peripheral blood was collected from a healthy donor via the vein. PBMCs were isolated using Ficoll lymphocyte separation medium and allowed to adhere and grow for two hours. Suspended cells, primarily T cells, were then collected. Three groups of K4D5 / CD28-CD19 CAR lentiviral vectors with different affinities were added to each group. A simple CD19 CAR without the enhanced receptor was also added to a separate group of T cells as a control. Lentivirus was added at an MOI (number of lentiviruses / number of cells) of 2, and the enhanced CAR-T cells were cultured and expanded.

[0215] Functional detection of K4D5 / CD28 by costimulation of OKT3 / HER2 proteins To verify the function of K4D5 / CD28, we first co-stimulated T cells with OKT3 and HER2 protein, and then detected cytokine secretion in an ELISA experiment. HER2 protein and OKT3 protein were both coated on a 96-well plate, and 1 × 10 T cells from each group were cultured. 5The cells were seeded into a 96-well plate at a density of 100 cells / well. After 48 hours of culture, the culture supernatants were collected and subjected to ELISA. The results showed that the IFN-γ secreted by the three groups of K4D5 / CD28-T cells and K4D5 / CD28-CAR-T cells with different affinities was significantly higher than that secreted by CAR-T cells without the enhanced receptor. However, when comparing K4D5 / CD28-CAR-T cells with different affinities, the stronger the affinity, the greater the IFN-γ secretion. Similar results were obtained for IL-2 detection. The results of the OKT3 / HER2 protein co-stimulation experiment showed that T cells expressing K4D5 / CD28 can effectively respond to CD3 activation signals, and when the CD3 signal is weak, K4D5 can effectively strengthen the activation signal by binding to HER2 protein, and further enhance T cell function. This enhancing effect is positively correlated with the affinity of HER2 protein, i.e., the stronger the affinity, the stronger the enhancing effect.

[0216] Detection of activation function of K4D5 / CD28-reinforced receptor-transfected CAR-T cells To verify the effect of K4D5 / CD28 on CAR-T cells, we first constructed a CD19-expressing J82-CD19 tumor cell line. CD19-carrying lentivirus was added to J82 cells at an MOI of 5, and CD19 expression was detected by flow cytometry 72 hours later. Flow cytometry results showed that 90% of J82 cells expressed CD19, indicating successful cell construction.

[0217] To verify the function of K4D5 / CD28, the inventors used a co-culture method with tumor cells and T cells to detect cytokine secretion by T cells. Results showed that IFN-γ secretion after co-culture of three groups of K4D5 / CD28-CAR-T cells with different affinities with J82-CD19 tumor cells exceeded that of CAR-T cells not transfected with the enhanced receptor. However, when comparing the three groups of K4D5 / CD28-CAR-T cells with different affinities, IFN-γ secretion increased with decreasing affinity. The results for IL-2 detection were generally consistent. In vitro experiments demonstrated that K4D5 / CD28 could effectively enhance the tumor-killing effect of CAR-T cells, but this enhancement was negatively correlated with the affinity of K4D5.

[0218] Example 5. Enhanced Receptor-Anti-HER2 Monoclonal Antibody / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of an anti-HER2 monoclonal antibody (abbreviated as aHER2mAb) and an intracellular domain consisting of 41BBz. The Fab domain of a HER2 antibody is typically designated K4D5. The enhanced receptor of this embodiment is designated aHER2mAb / 41BBZ, K4D5 / 41BBZ, or ER (Enhanced Receptor). The difference between the enhanced receptor of this example and that of Example 3 is the costimulatory molecule in the intracellular domain.

[0219] Amino acid sequences of three anti-HER2 antibodies, K4D5, with different affinities See Example 3.

[0220] Construction of three K4D5 / 41BBZ and K4D5 / 41BBZ-CD19 CAR lentiviral expression plasmids Three K4D5 / 41BBZ and K4D5 / 41BBZ-CD19 CAR lentiviral expression plasmids were constructed. K4D5 / 41BBZ and CD19 CAR were expressed as unfused vectors.

[0221] Preparation of K4D5 / 41BBZ and K4D5 / 41BBZ-CD19 CAR lentiviral vectors See Example 3 for the preparation process.

[0222] Preparation of CAR-T cells transfected with K4D5 / 41BBZ-enhanced receptor See Example 3 for the preparation process.

[0223] Functional detection of K4D5 / 41BBZ by costimulation of OKT3 / HER2 proteins For the experimental design, see Example 3. The experimental results show that T cells expressing K4D5 / 41BBZ can effectively respond to CD3 activation signals, and when the CD3 signal is weak, K4D5 can effectively strengthen the activation signal by binding to HER2 protein, thereby enhancing T cell function, and this enhancing effect is positively correlated with the affinity of HER2 protein, i.e., the stronger the affinity, the stronger the enhancing effect.

[0224] Detection of activation function of CAR-T cells transfected with K4D5 / 41BBZ-enhanced receptor For the experimental design, see Example 3. The results of the in vitro experiment showed that K4D5 / 41BBZ could effectively enhance the tumor-killing effect of CAR-T cells, and this enhancement was negatively correlated with the affinity of K4D5.

[0225] Example 6. Enhanced Receptor - Example Concerning Anti-HER2 Monoclonal Antibody / ICOS The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of an anti-HER2 monoclonal antibody (abbreviated as aHER2mAb) and an intracellular domain consisting of ICOS. The Fab domain of the HER2 antibody is designated K4D5. The enhanced receptor of this embodiment is designated aHER2mAb / ICOS, K4D5 / ICOS, or ER (Enhanced Receptor). The difference between the enhanced receptor of this example and that of Example 3 is the costimulatory molecule in the intracellular domain.

[0226] The experimental design was the same as in Example 3, except that the potentiating receptor was different.

[0227] The conclusions of the in vitro experiments were similar to those in Example 1, and ER-expressing CAR-T showed a stronger killing response against target cells compared with native, unmodified CAR-T.

[0228] The conclusions of the animal experiments were similar to those in Example 1, with ER-expressing CAR-T eliminating tumors more thoroughly than the CAR-T group not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the TIL density in the tumor tissue was higher than in the other groups (indicating that ER can help CAR-T cells better concentrate within tumors), and furthermore, the T cell (ER-T) group transfected with the enhanced receptor but not recognizing tumors was unable to kill tumors (indicating the safety of ER; i.e., ER will not independently kill PDL1-positive target cells unless T cells recognize target cells).

[0229] Example 7. Reinforcement Receptor - Example Concerning Anti-PDL1 Monoclonal Antibody / CD28 In this example, the inventors constructed and tested an enhanced receptor whose extracellular domain is an anti-PDL1 monoclonal antibody (abbreviated aPDL1mAb) and whose intracellular domain is CD28. In this embodiment, this enhanced receptor is referred to as aPDL1mAb / CD28 or ER (Enhanced Receptor). At the same time, a transmembrane-expressed aPDL1mAb protein is used as a control for this enhanced receptor.

[0230] Construction of lentiviral vectors of aPDL1mAb / CD28-enhanced receptor and transmembrane aPDL1mAb 1. A lentiviral vector expressing transmembrane aPDL1mAb without CD28 was constructed.

[0231] 2. The aPDL1mAb / CD28-enhanced receptor lentiviral vector was constructed. The construction process is described in Example 1.

[0232] Construction of other groups of lentiviral expression vectors Similar to the above steps, lentiviral vectors carrying the following gene sequences were constructed.

[0233] 1) An artificial TCR targeting the tumor target NY-ESO-1.

[0234] 2) CARs targeting CD19.

[0235] 4) Non-fused expression of an artificial TCR targeting PD1 / CD28 and the tumor target NY-ESO-1, termed ER-TCR.

[0236] 5) Non-fusion expression of CAR targeting PD1 / CD28 and CD19, termed ER-CAR.

[0237] Preparation of several populations of tumor-distinguishing T cells The following four types of tumor-discriminating T cells were prepared:

[0238] Preparation of TCR-T cells (NY-ESO-1-TCR-T or TCR-T) targeting tumor target NY-ESO-1 Patient leukocytes containing NY-ESO-1-positive, HLA-A:0201-type tumor cells were collected by apheresis. PBMCs were isolated using Ficoll lymphocyte separation medium, allowed to adhere for 2 hours, and floating cells, primarily T cells, were collected. A TCR lentiviral vector recognizing NY-ESO-1 was added at an MOI (number of lentiviruses / number of cells) of 1, and TCR-T cells were cultured and expanded. Flow cytometry revealed that the TCR expressed in 68.7% of the cells.

[0239] ER lentivirus was added to TCR-T at an MOI of 1 to obtain ER-TCR-T.

[0240] Preparation of CD19-targeting CAR-T cells (CD19CAR-T or CAR-T) and enhanced receptor-transfected CAR-T (ER-CAR-T) Patients' white blood cells were collected by apheresis, and PBMCs were isolated using Ficoll lymphocyte separation medium. After two hours of adherent growth, floating cells (mainly T cells) were collected. A CAR lentiviral vector that recognizes CD19 was added at an MOI (multiple infections per cell) of 1, and CAR-T cells were cultured and expanded.

[0241] ER lentivirus was added to CAR-T at an MOI of 1 to obtain ER-CAR-T.

[0242] Preparation of fresh tumor tissue-derived TIL cells and enhanced receptor-transfected TILs (ER-TILs) Tumor tissue cells were isolated from 10 grams or more of a patient's tumor tissue using enzymatic digestion, and CD3-positive T cells, or TILs, were isolated and purified using CD3 magnetic beads. The remaining cells were cultured in adherent culture to obtain the patient's autologous tumor cells.

[0243] ER lentivirus was added to TILs at an MOI of 1 to obtain ER-TILs.

[0244] Preparation of neoantigen-reactive T cells and enhanced receptor-transfected neoT (ER-neoT) First, whole-exome or RNA transcriptome sequencing was performed on the patient's peripheral blood or surgically resected tumor tissue. Mutation sites were selected, and affinity peptide prediction was performed in combination with the patient's HLA type. Approximately 20 mutations were selected as neoantigens. The neoantigens were gene-synthesized and transcribed into RNA in vitro. Again, the patient's peripheral blood was collected by apheresis or directly, and PBMCs were isolated and adherently expanded for 2 hours. To promote DC differentiation and maturation, adherent monocytes were supplemented with relevant cytokines, and RNA was introduced into the cells by electroporation. Suspension cells, primarily T cells, were co-cultured with electroporated transformed DCs. CD137+ T cells were isolated and expanded to obtain neoantigen-reactive T cells (neoT).

[0245] ER lentivirus was added to neoT at an MOI of 1 to obtain ER-neoT.

[0246] In vitro functional testing of aPDL1mAb / CD28-enhanced receptor expression in TCR-T targeting NY-ESO-1 1) ELISA plate experiment To verify the effects of aPDL1mAb / CD28-reinforced receptor on normal T cells and TCR-T targeting NY-ESO-1, we first prepared ELISA plates coated with 0.5 μg of OKT3 (CD3 antibody) protein, 1 μg of PDL1-Fc region + 0.5 μg of OKT3 protein, or 1 μg of PDL1-Fc region + 0.1 μg of OKT3 protein. Then, the following types of T cells were added to each plate, and IFNγ (Figure 1) and IL2 (Figure 2) were detected 48 hours later.

[0247] NOTD are unmodified normal T cells.

[0248] PDL1-T are T cells bearing aPDL1mAb-CD28.

[0249] PD1-T are T cells that have PDL1-CD28.

[0250] PDL1-TCR-T are TCR-T cells that carry aPDL1mAb-CD28.

[0251] PD1-TCR-T is a TCR-T cell that has PD1-CD28.

[0252] Here, PDL1 is aPDL1mAb-CD28, and PD1 is PD1-CD28.

[0253] As can be seen from Figures 1 and 2, after T cells and TCR-T cells with the enhanced receptor aPDL1mAb encountered PDL1 protein, which suppresses T cell function (two groups: 1 microgram of PDL1-Fc region + 0.5 micrograms of OKT3 protein, and 1 microgram of PDL1-Fc region + 0.1 micrograms of OKT3 protein), they secreted higher levels of both IFNγ and IL2 than T cells and TCR-T cells without the enhanced receptor aPDL1mAb.

[0254] 2) Cell experiments To verify the effect of aPDL1mAb / CD28-reinforced receptor on TCR-T targeting NY-ESO-1, we first constructed a J82-NY-ESO-1-PDL1 tumor cell line with HLA type A:0201. Lentivirus carrying PDL1 and NY-ESO-1 was added to J82 cells at an MOI of 5. At 72 h, positive cells were selected by adding G418 and puromycin. Approximately two weeks later, PDL1 and NY-ESO-1 expression was detected by flow cytometry. Flow cytometry results indicated that a large number of J82 cells co-expressed PDL1 and NY-ESO-1, indicating successful cell construction.

[0255] To verify the function of ER, the inventors used the method of co-culturing tumor cells and T cells to detect CD107a expression and cytokine secretion in T cells. The results showed that CD107a expression in ER-TCR-T cells exceeded that of normal TCR-T. IFN-γ and IL-2 secretion also exceeded that of TCR-T. In vitro experiments showed that the tumor-killing ability of TCR-T cells was significantly improved after transfection with the ER switch.

[0256] To compare the function of the transmembrane aPDL1mAb, we co-cultured tumor cells with T cells to detect CD107a expression and cytokine secretion in T cells. The results showed that the expression of CD107a, IFN-γ, and IL-2 in aPDL1mAb-NY-ESO-1-TCR-T cells was similar to that in normal TCR-T cells, and did not promote or suppress activity.

[0257] In vitro functional testing of TILs expressing aPDL1mAb / CD28-enhanced receptor To verify the effect of ER on TILs, the inventors co-cultured tumor cells isolated from fresh tumor tissues of patients with TILs and cells for 24 hours, respectively, and detected the secretion of IFN-γ and IL-2 by ELISA. The experimental results showed that the killing ability of ER-TILs was significantly higher than that of TILs. In vitro experiments showed that the tumor-killing ability of TILs was significantly improved after transfection with the ER switch.

[0258] To compare the function of the transmembrane aPDL1mAb, we co-cultured tumor cells with T cells to detect CD107a expression and cytokine secretion in T cells. The results showed that the expression of CD107a, IFN-γ, and IL-2 in aPDL1mAb-TIL cells was similar to that in normal TILs, and did not promote or suppress activity.

[0259] In vitro functional studies of neoantigen-reactive T cells expressing aPDL1mAb / CD28-enhanced receptors To verify the effect of ER on neoT, we co-cultured tumor cells isolated from fresh tumor tissues of patients with neoT and ER-neoT cells for 24 hours, respectively, and detected the secretion of IFN-γ and IL-2 by ELISA. The results showed that the killing ability of ER-neoT was significantly higher than that of neoT. In vitro experiments showed that the tumor-killing ability of neoT was significantly improved after transfection with the ER switch.

[0260] To compare the function of the transmembrane aPDL1mAb, we co-cultured tumor cells with T cells to detect CD107a expression and cytokine secretion in T cells. The results showed that the expression of CD107a, IFN-γ, and IL-2 in aPDL1mAb-neoT cells was similar to that in normal neoT cells and did not promote or suppress their activity.

[0261] In vitro functional experiments of CAR-T cells expressing aPDL1mAb / CD28-reinforced receptor First, we constructed a CD19-expressing tumor cell line and co-cultured it with conventional CD19 CAR-T cells (not transfected with the enhanced receptor) and ER-CAR-T for 24 hours. IFN-γ and IL-2 secretion were detected by ELISA. The killing ability of ER-CAR-T was significantly higher than that of conventional CAR-T (not transfected with the enhanced receptor).

[0262] To compare the function of the transmembrane aPDL1mAb, the inventors used a co-culture method with tumor cells to detect CD107a expression and cytokine secretion in T cells. The results showed that the expression of CD107a, IFN-γ, and IL-2 in aPDL1mAb-CAR-T cells was similar to that of conventional CAR-T cells and did not promote or suppress activity.

[0263] Animal experiments of NY-ESO-1 TCR-T cells expressing aPDL1mAb / CD28-enhanced receptor Inoculation: 1×10 6 The tumor cells J82-NY-ESO-1-PDL1 were subcutaneously inoculated into NSG mice, and a blank control group received a subcutaneous injection of PBS (A0). Tumor formation began in the tumor cell-injected mice approximately two weeks later. Tumor size was measured on day 23, and 30 mice were recruited.

[0264] The results of animal experiments on the binding of ER and TCR-T are shown in Figure 3. In the figure notes, PDL1 / CD28 is the aPDL1mAb-CD28 enhanced receptor, and PD1 / CD28 is the PD1 / CD28 enhanced receptor.

[0265] Administration: PBS was injected into the tail vein of the blank control group (A0), and the mice inoculated with tumor cells were divided into five groups: non-administered PBS group (A1), non-modified normal T cell group (NOTD, A2), TCR-T cell group (A3), ER-T CRT cell group (A4), and PD1 / CD28-T CRT (A5). All administration methods were 1 × 10 7 The cells were injected intravenously into the tail vein.

[0266] After administration, the tumor size was measured and the condition of the mice was observed every 2 to 3 days, and tumor volume = 1 / 2 x major axis x minor axis x minor axis was observed continuously for 30 days.

[0267] Changes in tumor burden: The tumor volume of the tumor-bearing mice continued to increase in each group over the course of one week after administration. The trend was essentially the same in groups A1 and A2, with deaths occurring on day 40. In groups A3, A4, and A5, tumor shrinkage was detected beginning on day 10. In group A3, rebound began on day 15, but in groups A4 and A5, tumor shrinkage continued until it nearly disappeared.

[0268] It can be seen that TCR-T cells bearing the enhanced receptor PD1 / CD28 or aPDL1mAb / CD28 clearly have stronger suppressive activity against tumors than TCR-T cells without the enhanced receptor.

[0269] Changes in total body mass of infused T cells: Of the three groups, one mouse was randomly selected from groups 3, 4, and 5 on the 10th day after administration, and tumor tissue was removed to isolate TILs. The total amount of re-administered cells was measured using flow cytometry, and it was found that the total amount of re-administered T cells in the tumors of group A5 was much higher than in the other groups.

[0270] Animal experiments with CAR-T cells expressing aPDL1mAb / CD28-reinforced receptor Inoculation: 1 x 10 expressing CD19 and PDL1 6 The tumor cells were subcutaneously inoculated into NSG mice, and a blank control group received a subcutaneous injection of PBS (A0). Tumor formation began in the tumor cell-injected mice approximately two weeks later. Tumor size was measured on day 23, and 30 mice were recruited.

[0271] Administration: PBS was injected into the tail vein of the blank control group (A0), and the mice inoculated with tumor cells were divided into five groups: non-administered PBS group (A1), T cell group (A2), ER-T cell group (A3), CAR-T cell group (A4), and ER-CAR-T cell group (A5). All administration methods were 1 × 10 7 The cells were injected intravenously into the tail vein.

[0272] After administration, the tumor size was measured and the condition of the mice was observed every 2 to 3 days, and tumor volume = 1 / 2 x major axis x minor axis x minor axis was observed continuously for 30 days.

[0273] Changes in tumor burden: The tumor volume of the tumor-bearing mice continued to increase in each group for one week after administration. The trend was basically the same in groups A1 to A3, with deaths occurring between the 30th and 40th day. In groups A4 and A5, tumor shrinkage was detected starting from the second week. In group A4, a rebound began between the 3rd and 4th week, but in group A5, tumor shrinkage continued until it almost disappeared.

[0274] Changes in total body mass of infused T cells: Of the three groups administered the drug, one mouse was randomly selected from groups 3, 4, and 5 on the 10th day after administration, and tumor tissue was collected to isolate and count T cells (i.e., TILs). The TIL density (i.e., the total number of TILs in the tumor tissue per unit weight) was calculated, and the results showed that the TIL density of the tumors in group A5 was far higher than that of the other groups.

[0275] Example 8. Reinforced Receptor - Example Concerning aPDL1mAb / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of an anti-PDL1 monoclonal antibody (abbreviated as aPDL1mAb) and an intracellular domain consisting of 41BBz. In this embodiment, this enhanced receptor is designated aPDL1mAb / 41BBz or ER (Enhanced Receptor). At the same time, transmembrane-expressed aPDL1mAb protein serves as a control for this enhanced receptor. The difference between the enhanced receptor in this example and that in Example 7 is the costimulatory molecule in the intracellular domain.

[0276] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0277] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0278] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0279] Example 9. Reinforcement Receptor - Example Concerning aPDL1mAb / ICOS The enhanced receptor constructed and tested by the inventors in this example has an anti-PDL1 monoclonal antibody (abbreviated aPDL1mAb) in its extracellular domain and ICOS in its intracellular domain. In this embodiment, this enhanced receptor is referred to as aPDL1mAb / ICOS or ER (Enhanced Receptor). At the same time, transmembrane-expressed aPDL1mAb protein serves as a control for this enhanced receptor. The difference between the enhanced receptor in this example and that in Example 7 is the costimulatory molecule in the intracellular domain.

[0280] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0281] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0282] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0283] Example 10. Example relating to the reinforcement receptor - NKG2D / CD28 The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of NKG2D and an intracellular domain of CD28. In this embodiment, this enhanced receptor is referred to as NKG2D / CD28 or ER (Enhanced Receptor). At the same time, the transmembrane-expressed NKG2D protein is used as a control for this enhanced receptor.

[0284] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0285] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0286] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0287] Example 11. Example relating to the potentiating receptor - NKG2D / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of NKG2D and an intracellular domain of 41BBz. In this embodiment, this enhanced receptor is designated NKG2D / 41BBz or ER (Enhanced Receptor). At the same time, transmembrane-expressed NKG2D protein serves as a control for this enhanced receptor. The enhanced receptor in this example differs from the enhanced receptor in Example 10 in the costimulatory molecule in the intracellular domain.

[0288] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0289] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0290] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0291] Example 12. Example relating to the potentiating receptor - NKG2D / ICOS The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of NKG2D and an intracellular domain of ICOS. In this embodiment, this enhanced receptor is designated NKG2D / ICOS or ER (Enhanced Receptor). At the same time, transmembrane-expressed NKG2D protein serves as a control for this enhanced receptor. The enhanced receptor in this example differs from the enhanced receptor in Example 10 in the costimulatory molecule in the intracellular domain.

[0292] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0293] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0294] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0295] Example 13. Enhanced Receptor - Anti-CD47 Monoclonal Antibody / CD28 Example The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of an anti-CD47 monoclonal antibody (abbreviated aCD47mAb) and an intracellular domain consisting of CD28. In this embodiment, this enhanced receptor is referred to as aCD47mAb / CD28 or ER (Enhanced Receptor). At the same time, a transmembrane-expressed aCD47mAb protein is used as a control for this enhanced receptor.

[0296] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0297] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0298] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0299] Example 14. Example relating to enhanced receptor-aCD47mAb / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of aCD47mAb and an intracellular domain consisting of 41BBz. In this embodiment, this enhanced receptor is designated aCD47mAb / 41BBz or ER (Enhanced Receptor). At the same time, transmembrane-expressed SIRPα protein is used as a control for this enhanced receptor. The difference between the enhanced receptor in this example and that in Example 13 is the costimulatory molecule in the intracellular domain.

[0300] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0301] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0302] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0303] Example 15. Example relating to the enhanced receptor - aCD47mAb / ICOS The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain consisting of aCD47mAb and an intracellular domain consisting of ICOS. In this embodiment, this enhanced receptor is referred to as aCD47mAb / ICOS or ER (Enhanced Receptor). At the same time, the transmembrane-expressed SIRPα protein is used as a control for this enhanced receptor. The difference between the enhanced receptor in this example and that in Example 13 is the costimulatory molecule in the intracellular domain.

[0304] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0305] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0306] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0307] Example 16. Example relating to the potentiating receptor - SIRPα / CD28 The enhanced receptor constructed by the inventors in this example has an extracellular domain of SIRPα and an intracellular domain of CD28. In this embodiment, this enhanced receptor is referred to as SIRPα / CD28 or ER (Enhanced Receptor). At the same time, the transmembrane-expressed SIRPα protein is used as a control for this enhanced receptor.

[0308] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0309] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0310] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0311] Example 17. Example relating to potentiating receptor - SIRPα / 41BBz The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of SIRPα and an intracellular domain of 41BBz. In this embodiment, this enhanced receptor is designated SIRPα / 41BBz or ER (Enhanced Receptor). At the same time, transmembrane-expressed SIRPα protein is used as a control for this enhanced receptor. The difference between the enhanced receptor in this example and that in Example 16 is the costimulatory molecule in the intracellular domain.

[0312] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0313] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0314] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

[0315] Example 18. Example relating to potentiator receptor - SIRPα / ICOS The enhanced receptor constructed and tested by the inventors in this example has an extracellular domain of SIRPα and an intracellular domain of ICOS. In this embodiment, this enhanced receptor is referred to as SIRPα / ICOS or ER (Enhanced Receptor). At the same time, transmembrane-expressed SIRPα protein is used as a control for this enhanced receptor. The difference between the enhanced receptor in this example and the enhanced receptor in Example 16 is the costimulatory molecule in the intracellular domain.

[0316] The experimental design is the same as in Example 7, except that the potentiating receptor is different.

[0317] The conclusions of the in vitro experiments were similar to those in Example 7, with ER-expressing TCR-T, CAR-T, TIL, and neoT all exhibiting stronger killing responses against target cells than the native, unmodified TCR-T, CAR-T, TIL, and neoT, respectively.

[0318] The conclusions of the animal experiments were similar to those in Example 7: TCR-T and CAR-T expressing ER eliminated tumors more thoroughly than TCR-T and CAR-T groups that were not transfected with the enhanced receptor (indicating the activity-enhancing properties of ER), the density of TILs in tumor tissue was higher than in the other groups (indicating that ER can help TCR-T and CAR-T cells better concentrate within tumors), and furthermore, the group of T cells that were transfected with the enhanced receptor but did not recognize tumors (ER-T) failed to kill tumors (indicating the safety of ER; i.e., if T cells do not recognize target cells, ER will not independently kill PDL1-positive target cells).

Claims

1. An enhanced receptor that improves the function of an immune cell, the enhanced receptor being a transmembrane protein expressed on the surface of an immune cell and comprising an extracellular domain (ECD) and an intracellular domain (ICD), An enhanced receptor, wherein the extracellular domain (ECD) is an anti-PDL1 monoclonal antibody, and the intracellular domain (ICD) comprises the intracellular domain of a costimulatory molecule that induces an immune cell activation signal, and does not comprise CD3ζ.

2. The enhanced receptor of claim 1, wherein the immune cell is any one of a T cell, an NK cell, a B cell, or a macrophage.

3. The enhanced receptor of claim 1, wherein the costimulatory molecule is selected from CD28, 41BBz, or ICOS.

4. The costimulatory molecule is CD28, and the sequence of the corresponding potentiating receptor is SEQ ID NO: 4; The costimulatory molecule is 41BBz and the sequence of the corresponding potentiating receptor is SEQ ID NO: 5; or The costimulatory molecule is ICOS, and the sequence of the corresponding potentiating receptor is SEQ ID NO:

6. The reinforcement receptor according to claim 3 .

5. The enhanced receptor of any one of claims 1 to 4, characterized in that the ECD can bind to a target cell of the immune cell, and the target cell of the immune cell is a cell harmful to the human body.

6. The enhanced receptor of claim 5, wherein the target cell is a tumor cell.

7. The enhanced receptor of claim 5, wherein the target cells are non-tumor cells.

8. The enhanced receptor of claim 7, wherein the target cell is a virus or a pathogen.

9. The enhanced receptor described in any one of claims 1 to 4, characterized in that the ECD can bind to a target cell of the immune cell, and the target cell of the immune cell is a cell that is harmless to the human body or harmless in the short term.

10. 10. The enhanced receptor of claim 9, wherein the target cells are selected from natural human cells or treated or irradiated non-human cells.

11. The enhanced receptor of claim 10, wherein the target cell is a B cell, a T cell, a NK cell, or a macrophage.

12. The enhanced receptor according to claim 10, wherein the target cell is K562, NK92 or KHYG-1.

13. (a) a reinforced receptor according to any one of claims 1 to 12, and (b) T cell receptor (TCR) or CAR (chimeric antigen receptor) A composition comprising: A composition in which the TCR is a naturally occurring unmodified or an artificially modified genetically engineered TCR.

14. 14. A genetically engineered T cell loaded with the composition of claim 13, wherein the T cell expresses a natural or artificial TCR or CAR and simultaneously transmembrane-expresses an enhanced receptor of any one of claims 1 to 12.

15. The genetically engineered T cell of claim 14, wherein the immune cell activation signal is activated by binding of the ECD of the enhanced receptor described in any one of claims 1 to 12 with a membrane protein of a target cell, thereby activating the function of the T cell and thereby enabling the T cell to resist the suppression of the target cell microenvironment in the T cell.

16. The genetically engineered T cell of claim 15, wherein the suppression of the target cell microenvironment in the T cell is from PDL1 or PDL2.

17. The genetically engineered T cells are: a) the T cells are a single clone characterized by a natural or artificial TCR or CAR capable of recognizing only a single target, or are a mixture of multiple T cell clones capable of recognizing multiple targets; b) the T cells are derived from tumor-infiltrating T lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) and are positive or negative for markers selected from PD1, TIM3, CD137 and CD39; c) the T cells have dual recognition, i.e., a first target cell is recognized by a first natural or artificial TCR or CAR, and simultaneously a second target cell is recognized by a second natural or artificial TCR or CAR, the first target cell being a cell harmful to the human body, and the second target cell being a cell harmless to the human body or harmless in the short term; 17. The genetically engineered T cell of any one of claims 14 to 16, characterized by one or more of the following:

18. The genetically engineered T cells of claim 17, characterized in that the genetically engineered T cells have dual specificity, i.e., on the one hand, they can identify and kill the first target cells to achieve the purpose of disease treatment, and on the other hand, they can be activated by the second target cells to enhance the therapeutic effect.

19. The genetically engineered T cell of claim 17, wherein the first target cell is a tumor cell.

20. 18. The genetically engineered T cell of claim 17, wherein the first target cell is a non-tumor cell.

21. 21. The genetically engineered T cell of claim 20, wherein the first target cell is a virus or pathogen.

22. 18. The genetically engineered T cell of claim 17, wherein the second target cell is selected from a natural human cell or a treated or irradiated non-human cell.

23. 23. The genetically engineered T cell of claim 22, wherein the second target cell is a B cell, a T cell, a NK cell, or a macrophage.

24. The genetically engineered T cell of claim 22, wherein the second target cell is K562, NK92, or KHYG-1.

25. The genetically engineered T cell of any one of claims 14 to 24, further comprising a suicide switch.

26. A cell medicine for treating a tumor, comprising the genetically engineered T cell according to any one of claims 14 to 24.

Images