Immunotherapy compositions and methods

Abstract

The present invention provides universal immunotherapy compositions useful for targeted therapy of cancer and other immune disorders.

Description

[0001] This invention is a divisional application of a PCT patent application filed in China with Chinese patent application number 201780011853.7, entitled "Immunotherapy Composition and Method", and international filing date of February 16, 2017. Technical Field

[0002] This invention generally relates to universal immunotherapy compositions that can be used for targeted therapy of cancer and other immune disorders.

[0003] Related applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 295,867, filed February 16, 2016, the entire contents of which are incorporated herein by reference.

[0004] Government rights This invention was made with the support of the National Cancer Institute (CA185151-02) and the National Institutes of Health (DK105602-01). The government holds certain rights to this invention. Background Technology

[0005] Clinical trials have demonstrated that cancer immunotherapy can induce durable responses in patients with advanced cancer. One of the most successful cancer immunotherapies uses chimeric antigen receptor (CAR) T cells to treat B-cell leukemia and lymphoma. The chimera used to fight these cancers is a single-chain antibody specific for CD19, which is fused to CD28 (a T-cell costimulatory protein) and subsequently to CD3ζ (a T-cell receptor (TCR) signaling protein). T cells expressing this construct receive primary and secondary signals and generate a robust immune response against all CD19-expressing cells, including normalized B cells. To date, CAR T-cell therapy has only achieved modest success against certain solid tumors because it is very difficult to identify antigens uniquely expressed on tumors rather than on unconverted cells. Although we can target tumor antigens exclusively, the vast majority of mutations are individualized and unique for each patient. Therefore, generating TCRs or antibodies that recognize neotumor antigens in each patient would be difficult and costly. This invention addresses this problem. Summary of the Invention

[0006] In various forms, the present invention provides a tumor antigen-specific binding molecule, wherein the binding molecule includes a recognition domain linked to a protective domain. This recognition domain specifically binds to a secondary binding molecule. In some forms, the secondary binding molecule is located on a cell. The cell is, for example, a chimeric antigen receptor T cell (CART), a T lymphocyte, a B lymphocyte, or a natural killer cell.

[0007] The binding molecule is an antibody, affinity, aptamer, or T-cell receptor (TCR) multimer. The antibody is Fab or scFV. The TCR multimer is a tetramer.

[0008] In some cases, the protective domain includes a carrier domain linked to a protease-sensitive peptide, which is specific to an oncogenic protease. Alternatively, the protective domain is pH-sensitive. A recognition domain is linked to the protective domain, so that the recognition domain is exposed when the protective domain comes into contact with a protease or is at a specific pH.

[0009] The carrying domain is a polymer, such as PEG-diacrylate.

[0010] This recognition domain is for small molecules. In some cases, it is for polymers. For example, the recognition domain can be a naturally occurring inorganic or organic compound, a synthetic inorganic or organic compound, or a biomolecule. This biomolecule can be a drug, toxin, hormone, metal, cytokine, peptide, or nucleic acid. Exemplary recognition domains include amphetamine, benzodiazepine, benzoylecone, buprenorphine, opioids, phencyclohexidine, tricyclic antidepressants, dextromethorphan, fentanyl, metronidazole, methadone, oxycodone, tramadol, zolpidem, ketamine, methylquinolones, propoxyphene, or norketimine.

[0011] In other embodiments, the present invention provides a chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain capable of binding a recognition domain. The extracellular domain is an antibody, such as Fab or scFV. The transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain. The transmembrane domain includes CD28.

[0012] The CAR further includes one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signaling domain. These co-stimulatory molecules are, for example, CD28, 4-1BB, 4-1BBL, ICOS, or OX40.

[0013] In various states, this intracellular signal transduction domain contains the CD3ζ chain.

[0014] Also provided is a nucleic acid encoding a CAR according to the invention, a vector comprising the nucleic acid, and a cell containing the vector. The cell is a T cell, for example, a CD4 cell. + T cells and / or CD8 + T cells, regulatory T cells (Treg), or follicle-regulating T cells (TFR).

[0015] In other embodiments, the present invention provides a genetically engineered cell that expresses and loads the chimeric antigen receptor according to the present invention onto its cell surface membrane. This cell is a T cell, for example, a CD4 cell. + T cells and / or CD8 + T cells, regulatory T cells (Treg), or follicle-regulating T cells (TFR).

[0016] In other embodiments, the present invention provides a pharmaceutical composition containing a genetically engineered cell population according to the present invention.

[0017] In another embodiment, the present invention provides a system comprising a tumor antigen-specific binding molecule and a chimeric antigen receptor (CAR), wherein the binding molecule has a recognition domain linked to a protective domain, and the CAR has an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain specifically binding to the recognition domain. In some embodiments, the CAR is expressed on a cell. The cell is a T cell, for example, a CD4 cell. + T cells and / or CD8 + T cells, regulatory T cells (Treg), or follicle-regulating T cells (TFR).

[0018] In other embodiments, the present invention provides a system comprising a binding molecule having tumor antigen specificity and a chimeric antigen receptor (CAR), wherein the binding molecule comprises (i) a cleavable masking portion that inhibits the binding molecule to the tumor antigen and (ii) a recognition domain; and the chimeric antigen receptor (CAR) has an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain that can specifically bind to the recognition domain.

[0019] Highly binding molecules are antibodies, affinity molecules, aptamers, or T-cell receptor (TCR) multimers. The antibody is Fab or scFV. The TCR multimer is a tetramer.

[0020] The cleavable masking portion is a protease-susceptible peptide.

[0021] In other embodiments, the present invention provides a method for treating cancer in a subject with such need, comprising administering the binding molecule according to the invention to the subject in a first period and administering the CAR according to the invention to the subject in a second period.

[0022] In another embodiment, the present invention provides a method for treating cancer in a subject with such need, the method comprising administering to the subject, in a first period, a binding molecule having tumor antigen specificity, wherein the binding molecule comprises (i) a cleavable masking portion that inhibits the binding of the binding molecule to the tumor antigen and (ii) a recognition domain; and, in a second period, administering to the subject a chimeric antigen receptor (CAR), the CAR comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain that specifically binds to the recognition domain.

[0023] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Although the invention may be practiced using methods and materials similar to those disclosed herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including its definitions, shall prevail. Furthermore, the materials, methods, and embodiments disclosed herein are for illustrative purposes only and are not intended to be limiting.

[0024] Other features and advantages of the invention will become apparent from and are covered by the following description and claims. Attached Figure Description

[0025] Figure 1 Examples of protease-specific exposure strategies used to induce tumor-specific T cell responses are provided.

[0026] Figure 2 For example, universal CAR T cells for tumor-targeted immunotherapy can be used to target exposed antigens.

[0027] Figure 3 Give an example of pH-dependent exposure to antigens.

[0028] Figure 4 Give an example of a test used to define the secretion of proteases in tumors.

[0029] Figure 5 Give an example of a test used to define the secretion of proteases in tumors.

[0030] Figure 6This is a schematic representation of the BAT-CAR strategy. BAT-CARs are specific for synthetic antigens that couple with tumor-targeting antibodies. The synthetic antigen is protected from T cell recognition by a "shield" containing a protease-sensitive site. When the tumor-targeting antibody binds to the tumor, tumor-derived proteins degrade this shield. Therefore, the synthetic antigen becomes readily recognized by the BAT-CAR T cell and is subsequently activated to guide an immune response against the tumor. Generated using a schematic template freely available from http: / / wmv.servier.com / Powerpoint-image-bank. Figure 6 , Figure 7 , Figure 8 and Figure 10 .

[0031] Figure 7 This is a schematic diagram of an example of the BAT-CAR T cells and CAR module used in this patent.

[0032] Figure 8 Activation of S8C cell populations transduced with anti-FITC or anti-AQ CAR modules was performed. FACS analysis was conducted, with CD69 overexpression as the readout.

[0033] Figure 9 This is a schematic diagram showing the cleavable peptide structure, the strategy of coupling to the BAT-CAR platform, and the chemical structures of the cleavable peptide carrying anthraquinone-2-ester molecule (CP(AQ)) and the cleaved peptide carrying the same molecule (pCP(AQ)).

[0034] Figure 10 This is a schematic diagram used to verify the different CU / TTU combinations that integrate the CU with the TTU.

[0035] Figure 11 HER2+ cells were stained with TTU labeled with Alexa fluor 647. FACS analysis was performed using the Alexa fluor 647 signal as a reading after 30 minutes of incubation on ice.

[0036] Figure 12 The top panel shows HER2+ cell staining with CU*FITC-PEG2-C(AQ)-PEG24*A647±unlabeled TTU. FACS analysis, FITC readings. The bottom panel compares the signal intensity of two different HER2+ cell populations stained with the same CU*FITC-PEG2-C(AQ)-PEG24*A647±unlabeled TTU. FACS analysis, FITC signal as readings, at two time points: 30 minutes of incubation on ice (red) and 8 hours of incubation at 37°C / 5% CO2.

[0037] Figure 13 It is a sequence of peptides carrying anthraquinone (AQ) small molecules, cleaved peptides (pCP(AQ)), and cleavable peptides (CP(AQ)). Detailed Implementation

[0038] This invention relates to general immunotherapy systems, compositions, and methods for treating cancer and other immune disorders such as autoimmune diseases and graft-versus-host diseases.

[0039] One of the biggest obstacles to cancer immunotherapy is identifying antigens uniquely expressed on tumor tissue rather than on normal healthy tissue. This invention overcomes these challenges by utilizing several technological innovations to create an effective and universal CAR T-cell therapy system. Specifically, this invention provides a system reagent capable of detecting any antibody on a tumor and generating an immune response against that tumor without off-target effects.

[0040] In short, in various forms, the present invention consists of three basic components: (1) a tumor-targeting binding molecule, (2) a masked small molecule, and (3) CAR T cells specific to the small molecule. The mask is sensitive to tumor-secreted biomolecules (e.g., proteases, lipases, glycosidases) or to the pH that causes the small molecule to be "exposed".

[0041] Overall measurements are presented in a schematic diagram. Figure 1 In this process, a series of binary events determine whether CAR T cells are activated by the synthetic small molecule. These T cells, which are binary activated using chimeric antigen receptors (BAT-CAR), should be completely inert in the absence of the small molecule and activated only at the site of exposure to the small molecule. The systems and compositions of the present invention can be adapted to guide T cell responses against any solid tumor in a patient-specific manner.

[0042] Another aspect of the invention is that any chimeric intracellular antibody can be engineered to be stimulated by a small molecule. In other words, the fusion of a single-chain antibody with any intracellular receptor can produce a novel chimeric receptor. Therefore, administration of a small molecule recognized by the single-chain antibody can stimulate downstream effects within the target cell, effects specific to stimulation of the receptor by its natural ligand. In this invention, T-cell receptor signaling can be induced by administering a small molecule. In other words, administration of a small molecule recognized by a single-chain antibody fused to a T-cell signaling molecule (e.g., CD28 and CD3ζ, but not exclusively) results in a change in the markers of T cells, which represent T-cell receptor signaling. By creating a chimera of a small molecule bound to a single-chain antibody with any cellular receptor, specific biological consequences can be induced by administering a small molecule recognized by the single-chain antibody.

[0043] The reagents according to the invention specifically generate a T-cell-guided immune response within tumors lacking prior information about neoantigens. The tumor is targeted by a binding molecule for the antigen, which is tumor-enriched but not necessarily tumor-specific. This binding molecule is coupled to a pharmacologically inert small molecule. This small molecule serves as a target for universal CAR T cells engineered with an extracellular binding domain, and this extracellular binding domain is specific to the small molecule. This universal CAR T cell, referred to herein as “binary activated T cells using chimeric antigen receptors (BAT-CAR),” is completely inert in the absence of the small molecule. Systemic treatment of a patient with a masked small molecule conjugated with the binding molecule delivers the small molecule to the tumor, creating a unique target for the BAT-CAR. To prevent off-target activation of the BAT-CAR T cells, the small molecule is masked using a “triggering” polymer (e.g., an enzyme-sensitive polymer or a pH-sensitive polymer). When intact, the polymer prevents the small molecule from binding and activating the BAT-CAR T cells. However, the tumor locally secretes proteases that digest the protease-sensitive "trigger" polymer, thus exposing only the small molecule at the tumor site. In the case of pH-sensitive tumors, the slightly acidic pH microenvironment of the tumor, compared to the physiological pH, digests the pH-sensitive "trigger" polymer, thus exposing only the small molecule at the tumor site.

[0044] In an alternative approach, a triggering polymer is used to mask the binding site of the binding molecule, thus inhibiting its binding to the tumor antigen. Similar to the case using a masked small molecule, a tumor-specific protease or pH digests the non-polymer, thereby allowing the binding molecule to bind to the tumor. This small molecule binds to and activates BAT-CAR T cells.

[0045] Combined molecules The binding molecules according to the present invention have binding specificity for tumor antigens. Hereinafter, the binding molecules are also referred to as "tumor-targeting units." The binding molecules can bind to or otherwise associate with biological entities such as membrane components, cell surface receptors, antigens, etc. The specificity of the binding molecules allows the aggregate of molecules to be localized to specific target sites, such as tumors, disease sites, tissues, organs, cell types, etc.

[0046] In this text, the term "bind" or "binding" refers to the interaction between corresponding pairs of molecules or portions thereof, typically exhibiting mutual affinity or binding ability due to specific or non-specific binding or interaction, including but not limited to biochemical, physiological, and / or chemical interactions. "Biobinding" defines a type of interaction occurring between pairs of molecules, including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, etc. The term "binding partner" refers to a molecule capable of binding to a specific molecule. "Specific binding" refers to a molecule that binds to or recognizes a binding partner (or a limited number of binding partners) to a degree greater than that of another similar biological whole.

[0047] The binding molecules include, but are not limited to, antibody molecules, receptor ligands, peptides, haptens, aptamers, affinity molecules, T-cell receptor tetramers, and other targeting molecules known to those skilled in the art. For example, the binding molecule is expected to include nucleic acids, peptides, glycoproteins, carbohydrates, or lipids.

[0048] The binding molecule can be an antibody, and the term tends to include antibody fragments. For example, antibodies include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab', and F(ab')2 immunoglobulin fragments, synthetically stabilized Fv fragments such as single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), single variable domain (dAbs) microantibodies, combinatorial antibodies, and multivalent antibodies such as bisomatic antibodies and multi-scFv, and single-domain equivalents derived from camelids or engineered humans. Antibodies are produced using conventional immunoassays (e.g., polyclonal sera and hybridomas); or antibodies are prepared as recombinant fragments and generally expressed in *E. coli* after selection from phage display libraries or ribosome display libraries. Alternatively, a “combination antibody” containing a non-covalently linked VH and VL domains can be generated from a matrix template created from bacterial colonies that produce bisomatic antibodies. The term “antibody” also includes any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or be partially or entirely synthesized.

[0049] For example, the binding molecule is an affinity protein. An affinity protein is a small, highly stable protein engineered to display peptide loops, providing a high-affinity binding surface to specific target proteins. It is a low-molecular-weight protein of 12 to 14 kDa, derived from the family of cysteine ​​protease inhibitors. Affinity proteins are scaffolded, stable proteins based on the folding of cystatin proteins. They display two peptide loops and an N-terminal sequence that can be arranged arbitrarily, thereby binding to different target proteins with antibody-like high affinity and specificity. When this protein scaffold constrains the conformation that the peptide may take, the peptide is stabilized, thereby increasing binding affinity and specificity compared to the free peptide.

[0050] For example, the binding molecule can be a nucleic acid binding molecule (e.g., an aptamer) that binds to a cell type-specific marker. Typically, an aptamer is an oligonucleotide (e.g., DNA, RNA, or their analogues or derivatives) that binds to a specific target such as a polypeptide. Aptamers are short, synthetic, single-stranded oligonucleotides that specifically bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures that specifically bind to proteins or other cellular targets and are essentially chemical equivalents of antibodies. Aptamers are highly specific, relatively small in size, and do not induce immunity. Aptamers are typically selected using the bio-screening method known as SELEX (an exponentially enriched ligand systematic evolution technique) (Ellington et al. Nature. 1990; 346(6287): 818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., Curr Med Che 2011; 18(27):4206-14; this reference is incorporated herein by reference in its entirety). Methods for generating aptamers for any given target are well known in the field.

[0051] In some specific embodiments, the binding molecule may be a naturally occurring ligand or a synthetic ligand for a cell surface receptor.

[0052] In some specific embodiments, the target molecule is a carbohydrate. The carbohydrate can be natural or synthetic. The carbohydrate can be a derived natural carbohydrate. In some specific embodiments, the carbohydrate comprises monosaccharides or disaccharides, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannouronic acid, glucosamine, galactosamine, or neuraminic acid. In some specific embodiments, the carbohydrate is a polysaccharide, such as, but not limited to, amylopectin, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextrin, glycogen, starch, hydroxyethyl starch, carrageenan, polysaccharides, amylose, chitosan, N,O-carboxymethyl chitosan, alginate and alginic acid, starch, chitin, heparin, konjac gum, glucomannan, ochratoxin, heparin, hyaluronic acid, gel polysaccharides, and xanthan gum. In some specific embodiments, the carbohydrate is a sugar alcohol, such as, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol, or lactitol.

[0053] Tumor antigens are any cell surface molecules on tumor cells. Preferably, the tumor antigen distinguishes tumor cells from normal cells. By being uniquely expressed on tumor cells or overpresented within tumor cells compared to normal cells, tumor antigens can differentiate tumor cells from normal cells. Tumor antigens are polypeptides, peptides (e.g., MHC peptides), lipids, or carbohydrates.

[0054] Exemplary tumor antigens include, but are not limited to, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, myoderm, glial fibrillary acidic protein (GFAP), giant cystic lesion fluid protein (GCDFP-15), HMB-45 antigen, and protein melanin-A. (Melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, nerve-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimer form of pyruvate kinase M2 isoenzyme (tumor M2-PK), abnormal Ras protein, or abnormal p53 protein.

[0055] Identification domain The recognition domain serves as a target for universal CAR T cells. In this document, the recognition domain is also referred to as an "antigen small molecule." The recognition domain is linked to the binding domain in a linkage mode that does not interfere with the binding domain's ability to bind its ligand. The recognition domain is non-human. The recognition domain is directly linked to the binding molecule. Alternatively, the recognition domain is indirectly linked to the binding molecule (e.g., via the protective or carrier domain). The recognition domain is one or more (i.e., multiple) small molecules. The small molecule is synthetic or naturally occurring. The small molecule is biologically active or biologically inactive. In this document, the phrase "biologically active" refers to the characteristic of any substance that is active in a biological system and / or organism. For example, a substance is considered biologically active if it has a biological effect on the organism when administered to it. Preferably, the small molecule is non-immunogenic (i.e., preferably non-antigenic).

[0056] In this field, "small molecule" is generally understood to be an organic molecule with a molecular weight of less than about 5 kilodaltons (Kd). In some specific embodiments, the small molecule is less than about 4 Kd, about 3 Kd, about 2 Kd, or about 1 Kd. In some specific embodiments, the small molecule is less than about 800 Daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some specific embodiments, the small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol.

[0057] Exemplary small molecules that can be used to generate recognition domains include fluorescein isothiocyanate (FITC), anthraquinone-2-acid, amphetamine, benzodiazepine, benzoylecone, buprenorphine, opioids, phencyclohexadiidine, tricyclic antidepressants, dextromethorphan, fentanyl, methaqualone, methadone, oxycodone, tramadol, zolpidem, ketamine, methylquinone, propoxyphene, or norcide. Other exemplary small molecules used in this invention include those listed at http: / / www.randoxtoxicology.com / products / biochip-array and http: / / www.randoxtoxicology.com / products / biochip-array / doa-I.

[0058] Protected domain In various embodiments, the recognition domain is linked to a protection domain. Hereinafter, the protection domain is also referred to as a “shield.” The protection domain masks the recognition domain to prevent it from binding to and activating the BAT CAR. The protection domain is either enzyme-sensitive (e.g., a protease) or pH-sensitive, either wholly or locally. In some embodiments, the protection domain consists of one or more enzyme-sensitive sites (i.e., cleavable peptides) or one or more pH-sensitive sites. When an enzyme-sensitive site or pH-sensitive site is exposed to an enzyme such as a protease or a specific pH, the reagent is cleaved, thus exposing the recognition domain.

[0059] The protective domain can be made of any material or size, as long as it can serve as a carrier or platform for the recognition domain and / or the enzyme susceptibility site. Preferably, the material is non-immunogenic, i.e., it will not induce an immune response in the subject to whom the material will be administered.

[0060] The protected domain is composed, in whole or in part, of polymeric or non-polymeric materials.

[0061] The protective domain is linked to the binding molecule directly or indirectly via a carrier domain. Suitable carriers are known in the art. In a preferred embodiment, the carrier domain uses Akrivis ADAPT™ technology (see WO / 2014 / 100377, WO / 2012 / 177775, and US 20140186850, the contents of which are incorporated herein by reference in their entirety).

[0062] In some states, the protective domain consists of a masking peptide and a masking polymer. In other states, the protective domain further includes a detection domain.

[0063] A wide range of biodegradable and bionon-biodegradable biocompatible polymers are known in the fields of polymeric biomaterials, drug delivery, and tissue engineering (see, for example, Vacanti's U.S. Patents US 6,123,727, US 5,804,178, US 5,770,417, US 5,736,372, US 5,716,404; Shastri's U.S. Patents US 6,095,148, US 5,837,752; Anseth's U.S. Patent US 5,902,599; Mikos's U.S. Patents US 5,696,175, US 5,514,378, US 5,512,600; Barrera's U.S. Patent US 5,399,665; Domb's U.S. Patent US 5,019,379; Ron's U.S. Patent US 5,010,167; d'Amore's U.S. Patent US 5,010,167; and others). US 4,946,929; and US 4,806,621 and US 4,638,045 of Kohn; see also Langer, Acc. Chem Res. 33:94, 2000; Langer, J. Control Release 62:7, 1999; and Uhrich et al., Chem Rev. 99:3181, 1999; all of the above references are incorporated herein by reference.

[0064] Polymers include, but are not limited to: polyamides, polycarbonates, polyolefins, polyalkylene glycols, polyepoxides, polyalkylene terephthalates, polyvinyl alcohol, polyvinyl ethers, polyvinyl esters, polyhalogenated polyethylene, polyglycolic acid, polysiloxanes, polyurethanes and their copolymers, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylates and methacrylates, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate. Cellulose acetate, carboxyethyl cellulose, cellulose triacetate, sodium cellulose sulfate, polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polyisobutyl methacrylate, polyhexyl methacrylate, polyisodecyl methacrylate, polylauryl methacrylate, polyphenyl methacrylate, polymethyl methacrylate, polyisopropyl methacrylate, polyisobutyl methacrylate, polyoctadecyl methacrylate, polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, polyethylene phthalate, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, and polystyrene.

[0065] Examples of biodegradable polymers include ethylene-vinyl acetate, poly(meth)acrylic acid, polyamide, and their copolymers and mixtures.

[0066] Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho-esters), polyurethanes, polybutyric acid, polyvaleric acid, polycaprolactone, polyhydroxybutyrate, poly(lactide-co-glycolic acid), and poly(lactide-co-caprolactone); and natural polymers such as alginates and other polysaccharides, including dextran and cellulose, collagen, their chemical derivatives (substitution, addition, hydroxylation, oxidation, and other modifications of chemical groups such as alkyl and alkenyl groups, and other modifications made by those skilled in the art through conventional experiments), albumin and other hydrophilic proteins, corn protein and other alcohol-soluble gluten and hydrophobic proteins, their copolymers, and mixtures. Typically, these materials are degraded by enzymatic hydrolysis or by surface or overall erosion in water within the body. The aforementioned materials can be used alone, as physical mixtures (hybrids), or as copolymers. In some specific embodiments, the polymer is polyester, polyanhydride, polystyrene, polylactic acid, polyglycolic acid, and copolymers of lactic acid and glycolic acid, and mixtures thereof.

[0067] PVP is a non-ionizable hydrophilic polymer with an average molecular weight ranging from about 10,000 to 700,000 and the chemical formula (C.6H9NO)[n]. PVP is also known as poly[1-(2-oxo-1-pyrrolyl)ethylene], Povidone™, Polyvidone™, RP 143™, Kollidon™, Peregal ST™, Periston™, Plasdone™, Plasmosan™, Protagent™, Subtosan™, and Vinisil™. PVP is non-toxic, highly hygroscopic, and readily soluble in water or organic solvents.

[0068] Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a condensation polymer of ethylene oxide and water, with the general chemical formula HO(CH2CH2O)[n]H.

[0069] Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetate by replacing the acetate group with a hydroxyl group, and has the general formula (CH.sub.2CHOH)[n]. Most polyvinyl alcohols are soluble in water. PEG, PVA, and PVP are commercially available from chemical suppliers such as Sigma Chemical Company (St. Louis, Mo.).

[0070] In some specific embodiments, the particle may contain poly(lactic acid-co-glycolic acid) (PLGA).

[0071] The enzyme susceptibility site depends on the enzyme that is active in a specific disease state. For example, tumors are associated with a specific set of enzymes. If the disease state being analyzed is a tumor, the product is designed to have an enzyme susceptibility site that matches the enzymes expressed by the tumor or other disease tissues. Alternatively, the enzyme-specific site may be associated with an enzyme that is ubiquitous but absent in a specific disease state. In this example, the disease state would be associated with the absence of signaling from the relevant enzyme or a decrease in signal levels compared to a normal reference.

[0072] In this article, "enzyme" refers to any protein among the large number of proteins produced in living cells that accelerate or catalyze metabolic processes in an organism. Enzymes act on substrates. The substrate binds to the enzyme at a site called the active site, after which the enzyme-catalyzed reaction immediately occurs. Enzymes include, but are not limited to, proteases, glycosidases, lipases, heparinases, and phosphatases.

[0073] The enzyme's susceptibility site can be optimized to provide high catalytic activity (or other enzymatic activity) to a specific target enzyme, and to release an optimized detectable label for detection. A large number of other enzyme / substrate combinations associated with specific diseases or conditions are known to those skilled in the art and can be used according to the present invention.

[0074] In some cases, the enzyme susceptibility site is a peptide (also referred to herein as a "masking peptide"). This masking peptide comprises a cleavage site (i.e., a cleavable peptide) and one or more sites that can link the masking polymer to the binding molecule (either directly or via a carrier domain). Some methods allow the masking peptide to have the following formula: (Aa N1 ) p1 (Aa N2 ) p2 (Cleavable peptides) q1 (Aa C1 ) q Aa C2 In various states, when the amino acid sequence located on the N-terminal flanking side of this cleavable peptide has the general formula (Aa N1 ) p1 (Aa N2 ) p2 , in: p1 is an integer selected from 1 to 10; p2 is an integer selected from 2 to 20; Aa N1 Each time it appears, it is any amino acid, preferably selected independently from lysine, histidine, arginine, aspartic acid and glutamic acid; Aa N2Each occurrence is any amino acid, preferably selected independently from serine, threonine, asparagine, glutamine, and glycine; and Aa N2 Linked to the N-terminus of the cleavable peptide via peptide bonds.

[0075] In one specific embodiment, p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another specific embodiment, p1 is 1, 2, 3, 4, or 5. In another specific embodiment, p1 is 6, 7, 8, 9, or 10. In another specific embodiment, p1 is 1, 2, or 3. In another specific embodiment, p1 is 1 or 2. In another specific embodiment, p1 is 1.

[0076] In one specific embodiment, Aa N1 Each occurrence is any amino acid, preferably selected independently from lysine, histidine, and arginine. In one specific embodiment, Aa N1 Each occurrence is any amino acid, preferably selected independently from lysine and histidine. In one specific embodiment, Aa N1 Each occurrence is any amino acid, preferably selected independently from lysine and arginine. In one specific embodiment, Aa N1 Each occurrence is any amino acid, preferably selected independently from histidine and arginine. In one specific embodiment, Aa N1 It is lysine each time it appears. In one specific embodiment, Aa N1 It is histidine in each occurrence. In one specific embodiment, Aa N1 It appears as arginine each time.

[0077] In one specific embodiment, Aa N1 Each time it appears, it is independently selected from aspartic acid and glutamic acid. In one specific embodiment, Aa N1 It appears each time as aspartic acid. In one specific embodiment, a N1 It appears as glutamic acid each time.

[0078] In one specific embodiment, p1 is 1, and Aa N1 It is lysine. In one specific embodiment, p1 is 1, and Aa N1 It is histidine. In one specific embodiment, p1 is 1, and Aa N1 It is arginine. In one specific embodiment, p1 is 1, and Aa N1 It is aspartic acid. In one specific embodiment, p1 is 1, and Aa N1 It's glutamic acid.

[0079] In one specific embodiment, p1 is 2, and Aa N1Each time it appears, it is independently selected from lysine, histidine, and arginine. In one specific embodiment, Aa N1 Each time it appears, it is independently selected from lysine and histidine. In one specific embodiment, Aa N1 Each time it appears, it is independently selected from lysine and arginine. In one specific embodiment, Aa N1 Each time it appears, it is independently selected from histidine and arginine.

[0080] In one specific embodiment, p1 is 2, and Aa N1 Each time it appears, it is independently selected from aspartic acid and glutamic acid.

[0081] In one embodiment, p2 is an integer selected from 2 to 10. In one embodiment, p2 is an integer selected from 11 to 20. In one embodiment, p2 is an integer selected from 2 to 5. In one embodiment, p2 is an integer selected from 5 to 10. In one embodiment, p2 is an integer selected from 11 to 15. In one embodiment, p2 is an integer selected from 15 to 20. In one embodiment, p2 is 2, 3, or 4. In one embodiment, p2 is 2 or 3. In one embodiment, p2 is 2.

[0082] In one specific embodiment, Aa N2 Each occurrence is any amino acid, preferably selected independently from serine, threonine, and glycine. In one specific embodiment, Aa N2 Each occurrence is any amino acid, preferably selected independently from serine and glycine. In one specific embodiment, Aa N2 Each occurrence is any amino acid, preferably selected independently from threonine and glycine. In one specific embodiment, Aa N2 Each occurrence is any amino acid, preferably selected independently from serine and threonine. In one specific embodiment, Aa N2 It is serine each time it appears. In one specific embodiment, Aa N2 It is threonine each time it appears. In one specific embodiment, Aa N2 It appears as glycine each time.

[0083] In one specific embodiment, Aa N2 Each time it appears, it is independently selected from asparagine, glutamine, and glycine. In one specific embodiment, Aa N2 Each time it appears, it is independently selected from asparagine and glycine. In one specific embodiment, Aa N2 Each time it appears, it is independently selected from glutamine and glycine. In one specific embodiment, Aa N2 Each time it appears, it is independently selected from asparagine and glutamine. In one specific embodiment, Aa N2It appears as asparagine each time. In one specific embodiment, Aa N2 It was glutamine each time it appeared.

[0084] In various states, the amino acid sequence located on the C-terminal flanking side of this cleavable peptide has the general formula (Aa C1 ) q Aa C2 , in: q is an integer selected from 2 to 20; Aa C1 Each time it appears, it is any amino acid, preferably selected independently from serine, threonine, asparagine, glutamine, and glycine; Aa C2 It is any reactive group that can link the masking peptide to the masking polymer, binding domain, or carrier domain.

[0085] Aa C1 Linked to the C-terminus of the cleavable peptide via peptide bonds.

[0086] In one embodiment, q is an integer selected from 2 to 10. In one embodiment, q is an integer selected from 11 to 20. In one embodiment, q is an integer selected from 2 to 5. In one embodiment, q is an integer selected from 5 to 10. In one embodiment, q is an integer selected from 11 to 15. In one embodiment, q is an integer selected from 15 to 20. In one embodiment, q is 2, 3, or 4. In one embodiment, q is 2 or 3. In one embodiment, q is 2.

[0087] In one specific embodiment, Aa C1 Each occurrence is any amino acid, preferably selected independently from serine, threonine, and glycine. In one specific embodiment, Aa C1 Each occurrence is any amino acid, preferably selected independently from serine and glycine. In one specific embodiment, Aa C1 Each occurrence is any amino acid, preferably selected independently from threonine and glycine. In one specific embodiment, Aa C1 Each occurrence is any amino acid, preferably selected independently from serine and threonine. In one specific embodiment, Aa C1 It is serine each time it appears. In one specific embodiment, Aa C1 It is threonine each time it appears. In one specific embodiment, Aa C1 It appears as glycine each time.

[0088] In one specific embodiment, Aa C1Each time it appears, it is independently selected from asparagine, glutamine, and glycine. In one specific embodiment, Aa C1 Each time it appears, it is independently selected from asparagine and glycine. In one specific embodiment, Aa C1 Each time it appears, it is independently selected from glutamine and glycine. In one specific embodiment, Aa C1 Each time it appears, it is independently selected from asparagine and glutamine. In one specific embodiment, Aa C1 In each occurrence, it is asparagine. In one specific embodiment, Aa is glutamine in each occurrence.

[0089] Aa C2 It is any reactive group capable of linking the masking peptide to the masking polymer, binding domain, or carrying domain. For example, it is maleimide, iodoacetamide, thioester, NHS ester, imine ester, hydroxymethylphosphine, carbodiimide, anhydride, carbonate, aldehyde, glyoxal, haloacetyl, pyridyl disulfide, vinyl sulfone, isocyanate, carbonyl diimidazole, benzophenone, anthraquinone, psoralen compounds, aryl azides, or halogenated aryl groups. In one specific embodiment, Aa C2 It is a semi-luminescent aminoamide. Coupling chemistry is known in this field (see, for example, Chemistry of Protein Conjugation and Cross-Linking, Shan S. Wong, CRC Press 1991, the contents of which are incorporated herein by reference in their entirety).

[0090] q1 is an integer selected from 1 to 20. In one embodiment, q1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another embodiment, q1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, q1 is 6, 7, 8, 9, or 10. In yet another embodiment, q1 is 1, 2, 3, 4, or 5. In yet another embodiment, p1 is 1 or 2. In yet another embodiment, q1 is 1.

[0091] The cleavable peptide contains an enzyme-susceptible site that can be cleaved by enzymes secreted by the tumor. Enzymes secreted by the tumor are known in the art and include, for example, proteases, lipases, and glycosidases. Preferably, the enzyme-susceptible site is a protease-susceptible site. The protease is, for example, a cysteine ​​protease, a serine protease, and an aspartate protease or a threonine protease.

[0092] Cysteine ​​proteases include, for example, cathepsin B, cathepsin L, cathepsin H, cathepsin S, calpain, MMP-1, MMP-2, MMP-7, MMP-9, or MMP-14. Serine proteases include, for example, trypsin, hepsin, KLK6, KLK7, KLK8, matriptase, SLP1, TMPRSS3, or PRSS3 / medium trypsin. Aspartate proteases include, for example, cathepsin D. Threonine proteases include, for example, the 26S proteasome.

[0093] Typically, the cleavable peptide will be 150 residues or less, 100 residues or less, or 50 residues or less. The total length can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. The expected peptide length ranges from 4 to 50 residues, 5 to 50 residues, 6 to 50 residues, 7 to 50 residues, 7 to 25 residues, 4 to 20 residues, 5 to 20 residues, 6 to 20 residues, 7 to 20 residues, and 7 to 15 residues.

[0094] Exemplary cleavable peptides include peptides comprising the amino acid sequence PLGVRG, PRCGVPDV, PRCGNPDV, PRCGXPD (where "X" is any amino acid, preferably "V"), PRCGVPDL, PRCGVPDK, or GPICFFRLGK.

[0095] In a preferred embodiment, the masking peptide comprises the amino acid LysSerGly ProLeuGlyValArgGly SerSerCys. In another preferred embodiment, the masking peptide comprises the amino acid sequence LysSerGly. ProLeuGlyValArgGly SerSer semi-luminescent amino amide.

[0096] "Any amino acid" means 20 common amino acids, stereoisomers of 20 common amino acids (e.g., D-amino acids), and non-natural amino acids.

[0097] In this document, the 20 common amino acids and their abbreviations follow conventional usage. See *Immunology - A Synthesis* (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of these 20 common amino acids (e.g., D-amino acids) and non-natural amino acids such as α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-traditional amino acids may also be suitable components for the peptides of this invention. Examples of non-traditional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphorylated serine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imines (e.g., 4-hydroxyproline). In accordance with standard usage and convention, in other polypeptide symbols used in this article, the left-hand direction is the amino terminus direction, and the right-hand direction is the carboxyl terminus direction.

[0098] When used for peptides, the term "substantial consistency" means that when two peptide sequences are optimally aligned, such as using the default gap weight alignment via the GAP or BESTFIT procedure, the two peptide sequences have at least 80% sequence consistency, for example, at least 90% sequence consistency, at least 95% sequence consistency, or at least 99% sequence consistency.

[0099] In some specific embodiments, the differences in the positions of the inconsistent residues are conserved amino acid substitutions.

[0100] Conservative amino acid substitution refers to the exchangeability of residues with similar side chains. For example, amino acid groups with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; amino acid groups with aliphatic-hydroxy side chains include serine and threonine; amino acid groups with amide-containing side chains include asparagine and glutamine; amino acid groups with aromatic side chains include phenylalanine, tyrosine, and tryptophan; amino acid groups with basic side chains include lysine, arginine, and histidine; and amino acid groups with sulfur-containing side chains include cysteine ​​and methionine. Suitable conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0101] The detection domain contains one or more detectable markers. In this document, "detectable marker" is understood to be a molecule that is detectable and preferably quantifiable when present in a sample or in a subject. "Detectable marker" includes, but is not limited to, enzyme-catalyzed markers (e.g., alkaline phosphatase), fluorescent markers, radioactive markers, dense particle markers, chemiluminescent markers, bioluminescent markers, prosthetic group markers, fluorescently emitting metal atoms, radioactive isotopes, quantum dots, nanoparticles, high electron density reagents, haptens, or biotin. Those skilled in the art will understand that the specific detectable marker used in a particular method will be determined by factors such as, for example, the target to be detected, the sample to which the detection is performed (e.g., liquid or solid sample, in vitro or in vivo detection), and the device used for detection. Detectable markers such as fluorescent markers can be directly detected. Alternatively, the detectable label can be detected by contacting it with at least one additional reagent, wherein the at least one additional reagent is, for example, an enzyme substrate that produces color, fluorescence, or luminescence; or a reagent that binds a non-sterically hindered chalaza, such as an avidin-containing label for binding a biotinylated chalaza, or a nickel-containing chalaza for binding 6 times His. This reagent can be a directly detectable reagent such as a fluorescent or radioactive tag, or an indirectly detectable reagent such as an enzyme.

[0102] In this document, the terms "labeled" or "labeled" refer to the incorporation of a detectable label, for example, by incorporating a radiolabeled amino acid or attaching a polypeptide to a biotinylated moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity detectable by optical or calorimetric methods). In some cases, the label or label can also be a therapeutic agent. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used in this invention. Examples of labeling for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 1) Fluorescent labeling (e.g., F1TC, rhodamine, lanthanide compounds of phosphorus), enzymatic labeling (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary receptors (e.g., leucine zippered sequences, binding sites of secondary antibodies, metal-binding domains, and additional epitopes). In some specific embodiments, the labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0103] CAR According to the present invention, the CAR typically comprises at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain; and a transmembrane polypeptide comprising at least one intracellular signal transduction domain; thus, these polypeptides are combined together to form a chimeric antigen receptor.

[0104] In this document, the term "extracellular ligand-binding domain" is defined as a polypeptide capable of binding a ligand. Preferably, the extracellular ligand-binding domain will be able to bind to the "exposed" recognition domain. For example, the extracellular ligand-binding domain binds to small molecules that constitute the recognition domain.

[0105] Extracellular binding domains include, but are not limited to, antibody molecules, receptor ligands, peptides, haptens, aptamers, affinity molecules, T-cell receptor tetramers, and other targeting molecules known to those skilled in the art. For example, the extracellular binding domain is expected to include nucleic acids, peptides, glycoproteins, carbohydrates, or lipids.

[0106] The extracellular binding domain can be an antibody, and the term tends to include antibody fragments. For example, antibodies include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab', and F(ab')2 immunoglobulin fragments, synthetically stabilized Fv fragments such as single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), single variable domain (dAbs) microantibodies, combinatorial antibodies, and multivalent antibodies such as bisomatic antibodies and multi-scFv, and single-domain equivalents derived from camelids or engineered humans. Antibodies are produced using conventional immunoassays (e.g., polyclonal sera and hybridomas); or antibodies are prepared as recombinant fragments and generally expressed in *E. coli* after selection from phage display libraries or ribosome display libraries. Alternatively, a “combination antibody” containing a non-covalently linked VH and VL domains can be generated from a matrix template created from bacterial colonies that produce bisomatic antibodies. The term “antibody” also includes any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or be partially or entirely synthesized.

[0107] For example, this extracellular ligand-binding domain is an affinity peptide. Affinities are small, highly stable proteins engineered to display peptide loops, providing a high-affinity binding surface to specific target proteins. They are low-molecular-weight proteins of the cysteine ​​protease inhibitor family, derived from cystin, with a molecular weight of 12 to 14 kDa. Affinity peptides are scaffolded, stable proteins based on the folding of cystatin proteins. They display two peptide loops and an N-terminal sequence that can be arranged arbitrarily, thereby binding to different target proteins with antibody-like high affinity and specificity. When this protein scaffold constrains the conformation that the peptide may take, the peptide is stabilized, thereby increasing binding affinity and specificity compared to the free peptide.

[0108] For example, the extracellular ligand-binding domain can be a nucleic acid-binding molecule (e.g., an aptamer) that binds to a cell type-specific marker. Typically, aptamers are oligonucleotides (e.g., DNA, RNA, or their analogs or derivatives) that bind to specific targets such as peptides. Aptamers are short, synthetic, single-stranded oligonucleotides that specifically bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures that specifically bind to proteins or other cellular targets and are essentially chemical equivalents of antibodies. Ligands are highly specific, relatively small in size, and do not induce immunity. Aptamers are typically selected using a biological screening method known as SELEX (Spiritually Enriched Ligand Systematic Evolution Technique) (Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968): 505-510; Ni et al., Curr Med Chem 2011; 18(27):4206-14; the above references are incorporated herein by reference in their entirety). Methods for generating aptamers for any given target are well known in the field.

[0109] In some specific embodiments, the binding molecule may be a naturally occurring or synthetic ligand of a small molecule antigen.

[0110] In a preferred embodiment, the transmembrane domain further includes a stalk region located between the extracellular ligand-binding domain and the transmembrane domain. Hereinafter, the term "stalk region" generally means an oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. Specifically, the stalk region is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may contain up to 300 amino acids, preferably 10 to 100 amino acids, more preferably 25 to 50 amino acids, and most preferably 3 to 15 amino acids. The stalk region may be derived from the whole or a portion of a naturally occurring molecule, such as from the whole or a portion of the extracellular region of CD8, CD4, or CD28, or from the whole or a portion of an antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or it may be a fully synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of the human CD8α chain.

[0111] The signal transduction domain or intracellular signal transduction domain of the CAR of this invention is the cause of intracellular signal transduction after the extracellular ligand binding domain binds to the target site, leading to immune cell activation and an immune response. In other words, this signal transduction domain is the cause of at least one normal effector function of CAR-expressing immune cells. For example, the effector functions of T cells may be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "signal transduction domain" refers to a portion of a protein that transduces effector signaling functions and guides the cell to perform specialized functions.

[0112] The signal transduction domain comprises two distinct classes of cytoplasmic signaling sequences: signaling sequences that initiate antigen-dependent primary activation, and signaling sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence may include signaling motifs known as tyrosine-based activation motifs of immune receptors, such as ITAMs. ITAMs are well-defined signaling motifs found in the intracytoplasmic tails of various receptors that serve as binding sites for syk / zap70-type tyrosine kinases. Examples of ITAMs used in this invention may include non-limiting examples derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signal transduction domain of the CAR may comprise the CD3ζ signaling domain, or the intracytoplasmic domain of the FcεRIβ or γ chain. In another preferred embodiment, for example, the signaling is provided by co-stimulation of CD3ζ with CD28 and tumor necrosis factor receptors (TNFr) such as 4-1BB or OX40.

[0113] In certain specific embodiments, the intracellular signaling domain of the CAR of the present invention comprises co-stimulatory signaling molecules. In some specific embodiments, the intracellular signaling domain comprises 2, 3, 4 or more co-stimulatory molecules in tandem. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response.

[0114] "Costimulatory ligands" refer to molecules located on antigen-presenting cells that specifically bind homologous costimulatory molecules to T cells. In addition to providing primary signals such as those from the binding of the TCR / CD3 complex to MHC molecules carrying peptides, they provide other signals that mediate T cell responses, including but not limited to proliferation activation and differentiation. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM), lymphotoxin β receptors, 3 / TR6, ILT3, ILT4, agonists or antibodies binding to Toll ligand receptors, and ligands that specifically bind to B7-H3. In addition, costimulatory ligands also include antibodies that specifically bind to costimulatory molecules presented on T cells, such as, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0115] "Co-stimulatory molecules" refer to homologous binding partners located on T cells that bind to co-stimulatory ligands, thereby mediating co-stimulatory responses, such as, but not limited to, proliferation, through the cell. Co-stimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD-3ζ, CD27, CD28, CD8, 4-1BB (CD137), -1BBL OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NLG2C, B7-H3, and ligands that specifically bind to CD83. In another specific embodiment, the signal transduction domain is the TNFR-associated factor 2 (TRAF2) binding motif and the intracytoplasmic tail of a co-stimulatory TNFR family member. The intracytoplasmic tails of co-stimulated TNFR family members contain a TRAF2-binding motif composed of a major conserved motif (P / S / A)X(QE)E) or a minor motif (PXQXXD), where X is any amino acid. In response to receptor trimerization, TRAF proteins are recruited by the intracytoplasmic tails of various TNFRs.

[0116] Significant characteristics of suitable transmembrane peptides include the ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and the ability to interact together to guide immune cell responses against pre-defined target cells. The various transmembrane peptides comprising an extracellular ligand-binding domain and / or a signal transduction domain in the CARs of the present invention interact together upon binding to a target ligand to participate in signal transduction and induce an immune response. The transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0117] As used herein, the term "a subset" refers to any subset of a molecule as a shorter peptide. Alternatively, functional variants of the amino acid sequence of the polypeptide can be prepared by mutations in the DNA encoding the polypeptide. These variants or functional variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can also be made to achieve the final construct, provided that the final construct possesses the attracted activity, particularly exhibiting specific anti-target cell immune activity. The functionality of the CAR of the present invention within host cells can be detected in assays suitable for demonstrating the signal transduction potential of the CAR when binding to a specific target. These assays are available to those skilled in the art. For example, the assay allows the detection of signal transduction pathways triggered upon binding to a target, such as assays involving measurements of the resulting increase in released calcium ions, intracellular tyrosine phosphorylation, inositol phosphate reversal, or the production of interleukin (IL)2, interferon-γ, GM-CSF, IL-3, and IL-4.

[0118] cell Specific embodiments of the present invention include cells expressing a CAR (i.e., CARTS). These cells can be of any kind, including immune cells capable of expressing a CAR for cancer therapy or cells such as bacterial cells that cryptically encode an expression vector for the CAR. In this document, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. All these terms also include their progeny, which are any and all descendants. It should be understood that all progeny may not be consistent due to intentional or unintentional mutations. In the context of expressing different nucleic acid sequences, “host cell” refers to a eukaryotic cell capable of replicating the vector and / or expressing a heterologous gene encoded by the vector. The host cell can and has been used as a recipient of the vector. The host cell can be “transfected” or “transformed,” referring to the process by which exogenous nucleic acids are transferred into or introduced into the host cell. Transformed cells include primary test cells and their progeny. In this document, the terms “engineered” and “recombinant” cells or host cells tend to refer to cells whose interiors have been introduced with exogenous nucleic acid sequences such as vectors. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain the recombinant-introduced nucleic acids. In specific embodiments of the present invention, the host cell is a T cell, including helper T cells (Th), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), regulatory T cells (Treg), T follicular regulatory cells (TFR), NK cells, and NKT cells are also covered by the present invention.

[0119] Some vectors may employ control sequences that enable them to replicate and / or be expressed in prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions required to incubate all of the aforementioned host cells to maintain the cells and allow vector replication. The techniques and conditions that allow for the large-scale production of vectors, as well as those that allow for the production of nucleic acids encoded by the vectors and their homologous polypeptides, proteins, or peptides, should also be understood and appreciated.

[0120] The cell can be an autologous cell, a cell with the same gene, an allogeneic cell, and in some cases, even a xenogeneic cell.

[0121] In many cases, in research or other events where the interest lies in the cell's disappearance after its presence, if one wishes to terminate the treatment, the cell becomes a tumor cell, and one can expect to kill the modified CTL. For this purpose, one can provide the expression of certain gene products in which the modified cells can be killed under controlled conditions, such as induced suicide genes.

[0122] Target CARTS The present invention further includes CARTS modified to secrete one or more polypeptides. The polypeptide may be, for example, an antibody or a cytokine. Cytokines include, for example, IL-2.

[0123] Targeted CARTS have the advantage of simultaneously secreting peptides at target sites such as tumor sites, transplantation sites, or autoimmune sites.

[0124] A targeted CAR-T can be constructed by including a nucleic acid encoding the polypeptide of interest following the intracellular signaling domain. Preferably, an internal ribosome entry site (IRES) is present between the intracellular signaling domain and the polypeptide of interest. Those skilled in the art will appreciate that a polypeptide can be reconstructed by employing multiple tandem IRES sequences.

[0125] Introducing structures into cells The expression vector encoding the CAR can be introduced as one or more DNA molecules or structures, wherein at least one marker that will allow a host cell containing the structure to secrete it can be present.

[0126] This construct can be prepared via conventional pathways, in which the gene and regulatory regions can be isolated, and, as appropriate, ligated, cloned in a suitable cloning host, and analyzed by definition, sequencing, or other conventional methods. Specifically, using PCR, individual fragments comprising all or part of the functional units can be isolated, wherein one or more mutations can be introduced, as appropriate, using primer repair, ligation, in vitro mutagenesis, etc. Once completed and proven to have the suitable sequence, the construct can be introduced into the cell (i.e., T cells) by any conventional method. The construct can be integrated and encapsulated within a non-replicating, defective viral genome such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including retroviral or lentiviral vectors for infection or transfection into cells. If desired, the construct may include viral sequences for transfection. Alternatively, the construct can be introduced via fusion, electroporation, gene gun, transfection, lipid transfection, etc. The host cell can be grown and expanded in a culture prior to the introduction of the construct, followed by appropriate treatment to introduce and integrate the construct. Subsequently, the cells are expanded and screened using markers present in this structure. Several markers that can be successfully used include hprt, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0127] In some cases, target sites for homologous recombination may be available, where it is desirable for the construct to be integrated into a specific genome. For example, an endogenous gene may be knocked out and replaced (in the same genome or elsewhere) with a gene encoding the construct using known materials and methods in the field for homologous recombination. For homologous recombination, OMEGA vectors or O vectors may be used. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0128] This construct can be introduced as a single DNA molecule encoding at least the CAR and another gene for the visual pattern, or as a different DNA molecule having one or more genes. Other genes include, for example, genes encoding therapeutic molecules or suicide genes. The construct can be introduced simultaneously or sequentially, each with the same or different markers.

[0129] It is well known in the field that vectors for preparing DNA constructs and for transfection contain available elements such as bacterial or yeast origin of replication, optional and / or amplifiable markers, promoter / enhancer elements for expression in prokaryotes or eukaryotes, and many are commercially available.

[0130] How to use The reagents according to the invention can be used to treat cancer or other immune disorders, such as graft-versus-host disease or autoimmune disorders, in patients with this need. In another specific embodiment, the reagents of the invention can be used to manufacture medicines for treating cancer or other immune disorders, such as graft-versus-host disease or autoimmune disorders, in patients with this need.

[0131] The present invention relies on a method for treating a patient in need of such treatment, the method comprising at least one of the following steps: (a) providing a binding molecule having tumor antigen specificity, wherein the binding molecule comprises a non-recognition domain linked to a protective domain; (b) providing a chimeric antigen receptor cell (CART) comprising an intercellular signaling domain, a transmembrane domain, and a recognition domain specifically binding thereto; and administering the molecule to the patient.

[0132] The treatment can be remission, cure, or prevention. It can be part of autologous immunotherapy or allogeneic immunotherapy. Autologous means that the cells, cell lines, or cell populations used to treat the patient originate from the patient or from a human leukocyte antigen (HLA) compatible donor. Allogeneic means that the cells or cell populations used to treat the patient do not originate from the patient but from a donor.

[0133] This invention is particularly applicable to allogeneic immunotherapy, to the extent that it enables the conversion of typical donor-derived T cells into allogeneic reactive cells. This can be performed using a standard protocol and repeated as needed. The resulting modified T cells can be pooled and administered as an "off-the-shelf" therapeutic product to one or more patients.

[0134] Cells that can be used with the disclosed methods are shown above. The treatment can be used to treat patients diagnosed with cancer, autoimmune diseases, or graft-versus-host disease (GvHD). Treatable cancers include non-vascularized or grossly non-vascularized tumors as well as vascularized tumors. The cancer may comprise non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) or may comprise solid tumors. Types of cancer to be treated using the CAR of this invention include, but are not limited to, carcinoma, germ cell tumor, sarcoma, certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcoma, carcinoma, and melanoma. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0135] It can be a treatment in combination with one or more anticancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiotherapy.

[0136] According to a preferred embodiment of the invention, the treatment can be administered to a patient undergoing immunosuppressive therapy. Indeed, the invention preferably relies on cells or cell populations that have been made resistant to an immunosuppressant due to the inactivation of a gene encoding at least one immunosuppressant receptor. In this respect, the immunosuppressive therapy should facilitate the selection and expansion of these T cells in the patient.

[0137] In other specific embodiments, the cell composition of the present invention is administered to the patient in combination with (e.g., before, concurrently with, or after) bone marrow transplantation, T-cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH. In another specific embodiment, the cell composition of the present invention is administered after B-cell ablation therapy such as rituximab (Rituxan), an agent that reacts with CD20. For example, in one specific embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some specific embodiments, after transplantation, the subject receives a fusion of the expanded immune cells of the present invention. In other specific embodiments, the expanded cells are administered before or after surgery. The modified cells obtained by any of the methods disclosed herein can be used, in a specific manner according to the present invention, to combat host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in patients in need; therefore, within the scope of the present invention, is a method for combating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in patients in need, comprising treating the patient by administering an effective amount of modified cells containing inactivated TCRα and / or TCRβ genes to the patient.

[0138] Cellular drug delivery This invention is particularly applicable to autologous immunotherapy, to the extent that it enables the conversion of typical donor-derived T cells into allogeneic reactive cells. This can be performed according to a standard protocol and repeated as needed. The resulting modified T cells can be pooled and administered as an "off-the-shelf" therapeutic product to one or more patients.

[0139] Based on the characteristics of this cell, it can be introduced into a host organism, such as a mammal, via various routes. In specific embodiments, the cell can be introduced at a tumor site, while in alternative embodiments, the cell can atrophy the cancer or be modified to atrophy the cancer. The number of cells used will depend on various factors, the purpose of introduction, cell lifespan, and the protocol to be used, such as multiple routes of administration, cell proliferation capacity, and the stability of recombinant structures. The cell can be applied as a dispersion, typically injected at or near the site of interest. The cell can be in a physiologically acceptable medium.

[0140] In some specific embodiments, the cell is encapsulated to inhibit immune recognition and placed at the tumor site.

[0141] The cells can be administered as desired. Various protocols can be employed depending on the desired response, the route of administration, the cell lifespan, and the number of cells present. The number of cells administered will depend at least in part on the factors disclosed above.

[0142] Administration of cells or cell populations according to the invention can be accomplished by any convenient method, including via nebulized inhalation, injection, swallowing, infusion, implantation, or transplantation. The compositions disclosed herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intralymphaticly, or intraperitoneally. In one specific embodiment, the cell compositions of the invention are preferably administered intravenously.

[0143] Administration of cells or cell populations may consist of the following: 10 mg / kg body weight 4 Up to 10 9 Cells, preferably 10 per kilogram of body weight 5 Up to 10 6 The effective amount of cells includes all integer values ​​within these ranges. The cells or cell population can be administered in one or more doses. In another specific embodiment, the effective amount of cells is administered as a single dose. In another specific embodiment, the effective amount of cells is administered as more than one dose over a period of time. The timing of administration is within the judgment of the attending physician and depends on the patient's clinical condition. The cells or cell population can be obtained from any source, such as a blood bank or donor. Although individual variations are possible, determining the optimal range of effective amounts for a given cell type for a specific disease or condition is within the scope of knowledge in the art. An effective amount is defined as the amount that provides therapeutic or preventative benefit. The dosage will depend on the recipient's age, health, and weight, the type of concurrent treatment if concomitant treatment is present, the frequency of treatment, and the characteristics of the desired effect.

[0144] It should be understood that this system involves multiple variables, such as cellular response to ligands, expression efficiency, apparent secretion levels, activity of the expressed product, patient-specific needs that can change over time and with environmental factors, and the rate of cell viability loss as a result of cell loss or loss of expression activity levels in individual cells. Therefore, it is expected that, for each patient, even with a universal cell line to be administered to the entire population, the individualized appropriate dose will be monitored, and that this patient monitoring is a standard practice in the field.

[0145] Nucleic acid-based expression systems The CAR of this invention can be expressed from an expression vector. Recombinant techniques for producing these expression vectors are well known in the art.

[0146] carrier The term "vector" refers to a carrier nucleic acid molecule that can be inserted into a vector for introduction into a cell, where it replicates. The nucleic acid molecule can be "exogenous," meaning it is foreign to the cell into which the vector is to be introduced, or its sequence is homologous to sequences within the cell, but is generally not found in the host cell's nucleic acid matrix. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art have knowledge of constructing vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0147] The term "expression vector" refers to any type of gene construct containing nucleic acids encoding RNA that can be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribonucleases. Expression vectors may contain a large number of "control sequences," which are nucleic acid sequences that are operatively linked to the coding sequence in a particular host cell and are essential for transcription and may be needed for translation. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences used for other functions and as described below.

[0148] Promoters and enhancers A promoter is a control sequence, a region of a nucleic acid sequence, that controls the initiation and rate of transcription. Promoters may contain genetic elements at which regulatory proteins and molecules, such as RNA polymerases and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The phrases "operably located," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to the nucleic acid to control the control and / or initiation of expression of that sequence.

[0149] Promoters typically contain a sequence that functions to locate the site of RNA synthesis initiation. The most well-known example is the TATA box, but in some promoters lacking a TATA box, such as those for mammalian terminal deoxyribonucleoside transferase genes and SV40 late-stage genes, discrete elements covering the initiation site themselves help to fix the initiation site. Other promoter elements regulate the frequency of transcription initiation. Typically, these elements are located 30-110 bp upstream of the initiation site, but many promoters have also been shown to contain functional elements downstream of the initiation site. To bring the coding sequence "under promoter control," the 5' end of the transcription start site of the transcription reading frame is positioned "downstream" (i.e., the 3' end) of the selected promoter. This "upstream" promoter stimulates transcription of the DNA and promotes the expression of the encoded RNA.

[0150] The spacing between promoter elements is often flexible, so promoter function is preserved when these elements are reversed or moved relative to each other. In the tk promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not work synergistically with "enhancers," which are cis-regulatory sequences involved in transcriptional activation within the nucleic acid sequence.

[0151] Promoters can be naturally associated with a nucleic acid sequence and can be obtained by isolating a 5' primer non-coding sequence located upstream of the coding segment and / or exon. This promoter can be referred to as "endogenous". Similarly, enhancers can be naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, where the recombinant or heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in their natural environment. These promoters or enhancers can include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any viral or prokaryotic or eukaryotic cell, and the promoter or enhancer is not "naturally occurring", i.e., containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, the promoters most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (tip) promoter systems. In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, these sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR in connection with the compositions disclosed herein. TM(See US 4,683,202 and US 5,928,906, each incorporated herein by reference). Furthermore, it is anticipated that control sequences guiding the transcription and / or expression of sequences located in non-nuclear organelles such as mitochondria and chloroplasts may also be employed.

[0152] Naturally, it is important to employ promoters and / or enhancers that effectively guide the expression of DNA segments within the selected organelle, cell type, tissue, organ, or organism for expression. The field of molecular biology reveals the use of any commonly known combination of promoters, enhancers, and cell types in protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter employed can be constitutive, tissue-specific, inducible, and / or available under suitable conditions to guide high-level expression of the introduced DNA segment, for example, advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter can be xenogeneic or endogenous.

[0153] Furthermore, any promoter / enhancer combination can be used to drive expression. Using T3, T7, or SP6 cytoplasmic expression systems is another possible specific embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if a suitable bacterial polymerase is provided, either as part of a delivery complex or as another gene expression construct.

[0154] The methods for identifying tissue-specific promoters or elements and characterizing their activity are well known to those skilled in the art.

[0155] Specific initiation signals may also be required for the efficient translation of the coded sequence. These may include the ATG initiation codon or adjacent sequences. An endogenous translation control signal, including the ATG initiation codon, may be required. Those skilled in the art will be able to easily determine this and provide the necessary signal.

[0156] In certain embodiments of the present invention, the internal ribosome entry site (IRES) element is used to create multigene or polycistronic information, and these can be used in the present invention.

[0157] In certain embodiments of the present invention, the 2A self-cleaving peptide is used to create multi-gene or multi-cis-self information, and these can be used in the present invention.

[0158] Vectors may include multiple cloning sites (MCS), which are nucleic acid regions containing multiple restriction enzyme sites, any one of which can work synergistically with standard recombinant techniques to digest the vector. "Restriction enzyme digestion" refers to the catalytic cleavage of nucleic acids using enzymes that function only at specific locations within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The uses of such enzymes are widely understood by those skilled in the art. Restriction enzymes are often used to linearize or fragment vectors, cutting within the MCS to allow endogenous sequences to be ligated into the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may be contiguous or discontinuous. Techniques involving restriction enzymes and ligation reactions are well-known to those skilled in the art of recombinant technology.

[0159] Splice sites, termination signals, replication origins, and optional markers can also be used.

[0160] plasmid vector In some specific embodiments, plasmid vectors are intended to be used to transform host cells. Typically, a plasmid vector containing replicons and control sequences, derived from a species compatible with the host cells, is associated with these hosts. Generally, the vector carries a replication site and a marker sequence that provides phenotypic selection within the transformed cells. In non-limiting examples, derivatives of pBR322—a plasmid derived from the species *E. coli*—are often used to transform *E. coli*. pBR322 contains resistance genes for ampicillin and tetracycline, and thus provides an easy means of identifying transformed cells. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, for example, a promoter that can be used by the microbial organism for its own protein expression.

[0161] In addition, phage vectors containing replicons and control sequences that are compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, phage λ GEM.TM. 11 can be used to create recombinant phage vectors that can be used to transform host cells such as Escherichia coli LE392.

[0162] Other available plasmid vectors include the p1N vector (lnouye et al., 1985) and the pGEX vector, used to generate soluble glutathione S-transferase (GST) fusion proteins for subsequent purification, separation, or lysis. Other suitable fusion proteins are those containing galactosidase, ubiquitin, etc.

[0163] Bacterial host cells containing the expression vector, such as *E. coli*, are grown in any of a variety of suitable culture media, such as LB medium. As those skilled in the art will understand, expression of the recombinant protein in certain vectors can be achieved by contacting the host cells with agents specific to certain promoters, such as by adding IPTG to the medium or by switching the incubation to a higher temperature. After further culturing the bacteria, typically between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual culture medium.

[0164] Viral vector The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and to integrate into the host cell genome and stably and efficiently express viral genes has made them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Components of the present invention may be viral vectors encoding one or more of the CARs of the present invention. Non-limiting examples of viral vectors that can be used to deliver the nucleic acids of the present invention are disclosed below.

[0165] Adenovirus vector One specific method for delivering this nucleic acid involves the use of adenoviral expression vectors. Although adenoviruses are known to have a low capacity for integration into genomic DNA, this characteristic is offset by the highly efficient gene transfer provided by these vectors. "Adenoviral expression vector" means those structures comprising adenoviral sequences sufficient to perform the following functions: (a) encapsulation supporting the structure and (b) the final expression of a tissue- or cell-specific structure that has been cloned within it. The understanding of the genetic makeup or adenovirus (a 36 kb linear double-stranded DNA virus) allows for the substitution of large segments of adenoviral DNA with foreign sequences, up to 7 kb (Grunhaus and Horwitz, 1992).

[0166] AAV carrier The nucleic acid can be introduced into the cell using adenovirus-assisted transfection. Increased transfection efficiency has been reported in cell systems using adenovirus coupling systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). For use in the cells of this invention, adeno-associated virus (AAV) is an attractive vector system because of its high integration frequency and ability to infect non-dividing cells, making it suitable for delivering genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad range of infectious hosts (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the generation and use of rAAV vectors are disclosed in US 5,139,941 and US 4,797,368, which are incorporated herein by reference.

[0167] Retroviral vector Retroviruses can be used as delivery vectors because of their ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be encapsulated in specific cell lines (Miller, 1992).

[0168] To construct a retroviral vector, a nucleic acid (e.g., one encoding the desired sequence) is inserted into the viral genome to replace certain viral sequences, producing a virus with replication defects. To generate virions, encapsulation cell lines and encapsulation components containing the gag, pol, and env genes but without the LTR are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a specific cell line (e.g., via calcium phosphate precipitation) along with the retroviral LTR and encapsulation sequence, the encapsulation sequence encapsulates the RNA transcript of the recombinant plasmid into the viral particle, which is then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). Subsequently, the culture medium containing the recombinant retrovirus is collected and concentrated as needed for gene transfer. Retroviral vectors can infect various cell types. However, integration and stable expression require the allocation of host cells (Paskind et al., 1975).

[0169] Lentivirals are retroviruses that, in addition to the common gag, pol, and env genes, contain other genes with regulatory or structural functions. Lentiviral vectors are well-known in this field (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; US 6,013,516 and US 5,994,136). Some examples of lentiviruses include human immunodeficiency virus HIV-1 and HTV-2, and simian immunodeficiency virus SIV. Lentiviral vectors have been produced by multiple attenuation of the HTV pathogenicity gene, for example, by deleting the env, vif, vpr, vpu, and nef genes, making the vector biologically safe.

[0170] Recombinant lentiviral vectors can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and nucleic acid sequence expression. For example, recombinant lentiviruses can infect non-dividing cells by transfection of appropriate host cells with two or more vectors carrying encapsulation functions named gag, pol, and env, and rev and tat, as disclosed in US 5,994,136, which is incorporated herein by reference. The recombinant virus can be targeted by linking the envelope protein to an antibody or a specific ligand targeting a receptor for a specific cell type. By inserting the sequence of interest (including regulatory regions) along with another gene encoding a ligand for a receptor on a specific target cell, the viral vector becomes, for example, significantly target-specific.

[0171] Other viral vectors In this invention, other viral vectors can be used as vaccine constructs. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used. They provide several attractive features for use in a variety of mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0172] Using modified virus delivery The nucleic acid to be delivered can be encapsulated within an infectious virus engineered to express a specific binding ligand. The viral particle will then specifically bind to a homologous receptor on the target cell and deliver its contents to that cell. Based on the chemical modification of the retrovirus by adding lactose residues to the viral capsid, a novel pathway designed to allow specific targeting by retroviral vectors has been developed. This modification allows for specific stem cell infection via the sialic acid glycoprotein receptor.

[0173] Another approach to designing targeted recombinant retroviruses involves using biotinylated antibodies against retroviral encapsulation proteins and specific cellular receptors. This antibody was coupled via a biotinylated component using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility antigen complexes of classes I and II, they demonstrated in vitro infection of various human cells carrying surface antigens of viruses with tropism (Roux et al., 1989).

[0174] Vector delivery and cell transformation Appropriate methods for delivering nucleic acids for transfecting or transforming cells are known to those skilled in the art. These methods include, but are not limited to, the direct delivery of DNA, RNA, or mRNA, such as through in vitro transfection, direct delivery by injection, etc. Cells can be transformed stably or transiently by applying techniques known in the art.

[0175] In vitro transformation Methods for transfecting eukaryotic cells and tissues removed from an in vitro organism are known to those skilled in the art. Therefore, it is contemplated that such cells or tissues can be transfected in vitro using the nucleic acid removal method of the present invention. In certain cases, the transplanted cells or tissues can be placed in an organism. In a preferred embodiment, the nucleic acid is expressed in the implanted cells.

[0176] The reagent kit of the present invention Any composition disclosed herein may be included in a kit. In a non-limiting example, one or more cells for cell therapy and / or reagents containing a recombinant expression vector for generating one or more cells for cell therapy may be included in the kit. The kit components are provided in suitable container units.

[0177] Some components of this kit may be encapsulated in an aqueous medium or in lyophilized form. The container units of the kit will typically include at least one vial, test tube, flask, bottle, syringe, or other container unit containing the component, preferably in appropriate equal volumes. If the kit contains more than one component, it will typically also contain a second, third, or another additional container, which may contain the other component separately. However, various combinations of components may be contained within a single vial. The kit of the present invention will also typically include the component in strictly limited commercial specifications. These containers may include injection-molded or blow-molded plastic containers containing the desired vial.

[0178] When the components of the kit are provided as one or more liquid solutions, the liquid solution is an aqueous solution, and in particular, a sterile aqueous solution may be used. In some cases, the container unit itself may be a syringe, pipette, and / or other similar device from which the preparation may be applied to the site of infection in the body, injected into an animal, and / or even applied to and / or mixed with other components in the kit.

[0179] However, the components of this kit may be provided as dry powders. When the reagents and / or components are provided as dry powders, the powders can be reconstituted by adding an appropriate solvent. It is also anticipated that the solvent may be provided in a separate container unit. The kit may also include a second container unit containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0180] In specific embodiments of the invention, cells intended for use in cell therapy are provided in the kit, and in some cases, the cells are essentially the sole component of the kit. The kit may contain reagents and materials for preparing the desired cells. In specific embodiments, the reagents and materials include primers for amplifying the desired sequence, nucleotides, suitable buffers or buffering agents, salts, etc., and in some cases, the reagents include a vector and / or DNA encoding a CAR as disclosed herein and / or regulatory elements for the vector and / or DNA.

[0181] In a specific embodiment, the kit includes one or more devices suitable for extracting one or more samples from an individual. These devices may be syringes, scalpels, etc.

[0182] In some embodiments of the present invention, in addition to specific embodiments of cell therapy, the kit also includes a second cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit can be tailored to suit a specific cancer in an individual and includes each of the second cancer therapies for that individual.

[0183] combination therapy In certain specific embodiments of the invention, the method of the invention for clinical manifestation is used in combination with other agents, such as anticancer agents, that are effective in the treatment of proliferative diseases. "Anticancer" agents can negatively affect cancer in a subject by, for example, killing cancer cells, introducing apoptosis into cancer cells, reducing the rate of cancer cell growth, reducing the incidence or number of metastases, reducing tumor volume, inhibiting tumor growth, reducing blood supply to tumor cells or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or increasing the lifespan of cancer patients. More generally, these other compositions will be provided in combined amounts that effectively kill or inhibit cell proliferation. This process may involve simultaneously exposing the cancer cells to the expression construct and the agent or multiple factors. This can be achieved by exposing the cells to a single composition or a pharmaceutical formulation comprising two agents, or by simultaneously exposing the cells to two distinct compositions or formulations, wherein one composition includes the expression construct and the other includes the second agent.

[0184] Tumor cell resistance to chemotherapy and radiotherapy agents presents a major problem in clinical oncology. Currently, a goal of cancer research is to find ways to improve the efficacy of chemotherapy and radiotherapy by combining them with other therapies. In the context of this invention, this cell therapy is also expected to work synergistically with interventions such as chemotherapy, radiotherapy, immunotherapy, and pre-apoptotic or cell circulation regulators.

[0185] Alternatively, the therapy of the present invention can be administered before or after treatment with the other agent, with intervals ranging from minutes to weeks. In various specific embodiments where the other agent is administered to an individual alone in conjunction with the present invention, it is generally ensured that the effective period between each delivery does not result in drug expiration, and thus the agent and the therapy of the present invention will still exert a beneficial combined effect on the cell. In these examples, it is anticipated that the cell will be exposed to both treatments, with an interval between the two treatments of about 12 to 24 hours, more preferably about 6 to 12 hours. However, in some cases, it is desirable to significantly extend the duration of treatment, wherein the interval between each administration is from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0186] The treatment cycle is expected to be repeated as needed. It is also anticipated that various standard therapies and surgical interventions can be used in conjunction with the cell therapy of this invention.

[0187] Chemotherapy Cancer treatment also includes various combination therapies based on chemotherapy and radiotherapy. Combination chemotherapy includes, for example, albumin-bound paclitaxel, hexamethylmelamine, docetaxel, Herceptin, methotrexate, novantrone, zoledrone, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, daunomycin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, paclitaxel, gemcitabine, vinorelbine, farnesyltransferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analogues or diffraction variants of the foregoing substances and combinations thereof.

[0188] In specific embodiments, the chemotherapy used on the individual is synergistic with the present invention, for example, the chemotherapy is performed before, during and / or after administration of the present invention.

[0189] Radiation therapy Other factors that cause DNA damage and have been widely identified include gamma rays, X-rays, and / or radioactive isotopes delivered directly to tumor cells. Other forms of DNA damage, such as microwave and UV radiation, are also considered. Most likely, all of these factors have spectral damaging effects on DNA, precursor DNA, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from long-term (3 to 4 weeks) daily doses of 50 to 200 roentgens to single doses of 2000 to 6000 roentgens. Radioactive isotope doses vary considerably and depend on the isotope's half-life, the intensity and type of radiation, and the absorption rate by tumor cells.

[0190] In this text, when used with respect to cells, the terms "contact" and "exposure" are used to describe a process by which a therapeutic agent and a chemotherapeutic or radiotherapy agent are delivered to or placed in direct proximity to a target cell. To achieve cell killing or cell arrest, a combination of both agents is delivered to effectively kill the cell or prevent its division.

[0191] Immunotherapy Immunotherapy typically relies on the use of immune effector cells and molecules that target and destroy cancer cells. These immune effectors can be, for example, antibodies specific to certain markers on the surface of tumor cells. The antibody can be used alone as an effector of the therapy, or it can restore other cells to achieve a cytotoxic effect. The antibody can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used solely as a target. Alternatively, the effector can be a lymphocyte carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0192] Therefore, immunotherapies other than those disclosed herein can be used in conjunction with the cell therapies of this invention as part of a combination therapy. The general steps of the combination therapy are discussed below. Typically, tumor cells must carry certain markers that are target-resistant, i.e., not present on most other cells. Many tumor markers are present, and any of these tumor markers may be suitable for targeting in the context of this invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate-specific antigen, urinary tract tumor-associated antigen, embryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin, erb B, and pl55.

[0193] Gene In another specific embodiment, the second treatment is gene therapy, wherein the therapeutic polynucleotide is administered before, after, or simultaneously with the clinical embodiment of the invention. Various expression products are covered by this invention, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0194] surgery Approximately 60% of cancer patients will undergo some type of surgery, including preventative surgery, diagnostic or staging surgery, curative surgery, and remissionary surgery. Curative surgery is a cancer treatment that can be combined with other therapies such as the treatments described in this invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0195] Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs's surgery). Furthermore, this invention is anticipated to be synergistic with the removal of superficial cancerous tissue, precancerous tissue, or incidental amounts of normal tissue.

[0196] Once some or all of the cancer cells, tissue, or tumor has been removed, a cavity is formed in the body. Treatment can be administered by infusing, injecting, or applying another anticancer therapy to this site. This treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, or 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The dosage of these treatments can also be varied.

[0197] Other reagents Other agents are anticipated to be used in conjunction with this invention to improve therapeutic efficacy. These other agents include immunomodulators, agents affecting the upregulation of cell surface receptors and intercellular junctions, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of overproliferating cells to apoptosis-inducing agents. Immunomodulators include tumor necrosis factor; interferon α, interferon β, and interferon γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. Further, it is anticipated that upregulation of cell surface receptors or their ligands, such as Fas / Fas ligands, DR4, or DR5 / TRAIL, will enhance the apoptosis-inducing ability of this invention by establishing autocrine or paracrine effects on overproliferating cells. Increasing intercellular signaling by increasing the number of intercellular junctions will increase the anti-overproliferative effect on adjacent overproliferating cell populations. In other specific embodiments, cell growth or differentiation inhibitors may be used in conjunction with this invention to improve the anti-overproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Further consideration is given to agents that increase the sensitivity of overproliferating cells to apoptosis, such as antibody C225, which could be used in combination with the present invention to improve the efficacy of the treatment.

[0198] definition In this document, the term "biocompatible" refers to a substance that is non-toxic to cells. In some specific embodiments, a substance is considered "biocompatible" if its addition to cells in vivo does not induce inflammation and / or other negative in vivo effects. In some specific embodiments, a substance is considered "biocompatible" if its addition to cells in vitro or in vivo results in cell death of less than or equal to about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5%.

[0199] In this document, the term "biodegradable" refers to a substance that can be degraded under physiological conditions. In some specific embodiments, a biodegradable substance is a substance that is destroyed by cellular mechanisms. In some specific embodiments, a biodegradable substance is a substance that is destroyed by chemical processes. For example, the degradation of an exemplary biodegradable material requires the presence of an enzyme. Physiological conditions are insufficient for most (if not all) such biodegradable materials.

[0200] In this document, when the terms “related to,” “synergistic,” “linked,” “attached,” and “ligated” are used to describe two or more parts, it means that these parts are physically associated or connected to each other, either directly or via one or more additional parts acting as linkers, to form a sufficiently stable structure, thus maintaining physical association under conditions of use of the structure, such as physiological conditions. In some specific embodiments, the parts adhere to each other via one or more covalent bonds. In some specific embodiments, the parts adhere to each other by involving specific (rather than covalent) binding (e.g., streptavidin / avidin interaction, antibody / antigen interaction, etc.). In some specific embodiments, a sufficient amount of weak interaction provides sufficient stability to maintain the parts as physically associated.

[0201] In this text, when the terms "synergistic," "linked," and "adhesive" are used to refer to two or more parts, it means that these parts are associated or connected, either directly or via one or more additional parts acting as linkers, to form a sufficiently stable structure, thus maintaining physical association under conditions of use of the structure, such as physiological conditions. These parts typically adhere either through one or more covalent bonds or through mechanisms involving specific binding. Alternatively, sufficient weak interactions may provide enough stability to maintain the part as physically associated.

[0202] In this text, the term "bind" or "binding" refers to an interaction between pairs of molecules or parts thereof, which typically exhibit mutual affinity or binding capacity due to specific or non-specific binding or interaction, including but not limited to biochemical, physiological, and / or chemical effects. "Biobinding" defines a type of interaction occurring between pairs of molecules, including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, etc. The term "binding partner" refers to a molecule that can bind to a specific molecule. "Specific binding" refers to a molecule that binds to or recognizes a binding partner (or a limited number of binding partners) to a degree substantially greater than that of another similar biological whole.

[0203] [Example] Example 1: Masked small molecule functionalized tumor-targeting antibodies The objective of this embodiment is to construct a scaffold composed of a backbone polymer structure coupled with small molecules, which can be further modified with a protein-sensitive hydrogel polymer. PEG is an excellent choice for functionalizing this small molecule scaffold, as its primers stabilize biomolecules and are resistant to degradation, thereby facilitating the coupling chemical reaction. Any number of small molecules can be selected. This PEG / small molecule / protease-sensitive hydrogel polymer scaffold will be coupled with a tumor-targeting antibody.

[0204] Example 2: Masked small molecule functionalized tumor-targeting antibodies The purpose of this embodiment is to utilize Probody® CytomX technology, which uses protease-sensitive peptides to block the ability of tumor-targeting antibodies to recognize their antigens. In this strategy, it is envisioned that the small molecule is directly coupled to the Fc portion of the antibody or coupled to some other site besides the antigen-recognizing portion of the antibody. We will select the small molecule based on its pharmacological activity.

[0205] Example 3: Creation of pH-dependent polymers that expose small molecules only near tumors The general strategy for this technology is to develop peptides whose conformation changes in a pH-dependent manner. We plan to covalently link a small molecule antigen to this peptide. Under physiological conditions, the peptide will fold back onto itself to mask the small molecule. Under acidic conditions, the peptide will open, exposing the small molecule. The advantage of this strategy is that antibodies not bound to the tumor will mask the small molecule, thereby blocking inappropriate or unplanned activation of the immune system. The general strategy for linking this hydrogel polymer to a Phase I tumor-targeting antibody is suitable for linking this peptide to that antibody. As an alternative strategy, this pH-sensitive peptide can be directly injected into the tumor to generate an immune response.

[0206] Example 4: Engineered CAR T cells to guide their resistance against small molecules Materials and methods The aim of this experiment was to engineer CAR T cells that could be triggered by a small molecule directly coupled to a tumor-targeting antibody or embedded in a protease-sensitive polymer coupled to that antibody. The CAR T cells were engineered using a single-chain antibody that recognizes the small molecule. The chimera was constructed using a fusion of the CD8a leader sequence, CD8a hinge sequence, CD28, 4-IBB, and CO3ζ intracellular domain. Figure 7 The composition of the CAR module used in this paper is visualized.

[0207] Generation of CAR constructs This molecular design was inspired by the publication Sun et al, Breast Cancer Res, 16(3):R61 (2014), in which the anti-HER2 antibody was fused between the CD8a leader sequence and the CD8a hinge sequence, followed by CD28 and CD3ζ intracellular domains. In contrast, the 4-1BB intracellular domain was inserted between CD28 and CD3ζ. Furthermore, no linker or spacer was used.

[0208] Two single-chain antibodies were cloned into the construct: an anti-FITC antibody as revealed in Boder et al, Proc Natl Acad Sci US A.26;97(20): 10701-5, (2000), and an anti-anthraquinone-2-carboxylic acid designed by Randox Biosciences (Admore, Diamond Road, United Kingdom).

[0209] This CAR T is expected to react with any small molecule, as long as it is a suitable single-chain antibody.

[0210] Each construct was cloned using Gibson assembly and sequenced using a traditional sequence service (GENEWIZ, Inc.).

[0211] Generation of troponin retroviruses The retroviral vector we chose for proof-of-concept was based on the vector disclosed by Hoist et al. J. Gen. Virol. 88: 1708-1716 (2007) and was available via Addgene (plasmid #52107). In short, the CAR construct was inserted into an empty retroviral vector containing the MSCV's ψ sequence and a C-terminal GFP reporter gene, which was separated from the CAR sequence by a self-cleaving IRES module.

[0212] The tropism-dependent retrovirus was generated using the PLAT-E encapsulated cell line (Cell Biolabs, Inc.), as described by the manufacturer. In brief, PLAT-E cells were plated in DMEM, 10% fetal bovine serum (FCS), 1 μg / mL puromycin, 10 μg / mL blastomycin, penicillin, and streptomycin. The day before transfection, the cells were grown in 150 mm tissue culture plates (Falcon, #353025) in DMEM, 10% (FCS) until 70% confluence. On the day of transfection, 2.0 ml of Opti-MEM (ThermoFisher Scientific, #31985070) was mixed with 112.5 μl of Lipofectamine 2000 (Invitrogen, #11668500) and incubated at room temperature for 10 minutes. Then, 30 μg of plasmid was added, and the mixture was incubated at room temperature for another 15 minutes. Throughout the cell culture process, the Opti-MEM / lipofectamine 2000 / DNA solution was added dropwise using a helical motion. Cells were incubated at 37°C / 5% CO2 for 48 hours. Upon harvest, the supernatant was collected and filtered through a 0.45 μm polyethersulfone (PES) filter (CELLTREAT Scientific Products, #229749). The filtered solution was divided into two aliquots: one aliquot was immediately used for transduction of mouse T cells, and the other aliquot was further divided and stored at -80°C.

[0213] Transduction of mouse hybridoma T cells T cell activation upon antigen exposure was studied using mouse hybridoma T cells (58C) deprived of TCR α and β chains (Letourneur and Malissen, Eur J Immunol. Dec;19(12):2269-74, (1989)).

[0214] The mouse T cells were seeded in RPMI 10% FBS containing penicillin and streptomycin. On the day of transfection, 300,000 cells were seeded in the wells of a 6-well Costa plate (Corning, #3516). Viral supernatant containing 5 μg / ml polybrene (Santa Cruz Biotechnology, #sc-134220) was added to each well. After gently mixing the cells with the viral supernatant, the cells were centrifuged at 37°C and 800g for 1 hour in a Sorvall RT centrifuge (ThermoFisher, #EW-17705-10). After centrifugation, the viral supernatant was replaced with 2 ml of RPMI 10% FBS containing penicillin and streptomycin. The remaining small aliquot of viral supernatant was stored overnight at 4°C. 16 to 24 hours later, rotation infection was performed using 1 ml of the same viral supernatant previously stored at 4°C. The transduced cells were harvested, incubated at 37°C / 5% CO2 for 48 hours, and then classified to obtain GFP-positive cells.

[0215] Quantitative analysis of T cell activation as a function of CD69 surface expression At 37°C, the wells of a 24-well Falcon plate (Corning, #353047) were coated for 1 hour with 500 μl of a 10 μg / ml antigen solution in PBS. The antigen solution contained a peptide bound to the antigen molecule for anti-FITC CAR T cells: BSA-FITC (Life Technologies, #A23015), and a cleaved peptide bound to anthraquinone-2-ester for anti-AQ CART (pCP(AQ), SEQ ID NO: 1). As a mock experiment, each well involving CAR T cells was coated with BSA (Sigma-Aldrich, #A7906). After coating, 500,000 58C cells in 500 μl of RPMI 10% FBS containing penicillin and streptomycin were seeded in each well. The 58C cells were incubated at 37°C for 4 hours in the presence of their antigen before FACS analysis.

[0216] Prior to FACS analysis, 250 μl of cell solution was transferred to a 96-well Falcon plate with a round bottom (Corning, #353077). Each plate was centrifuged at 500 g for 3 minutes at 4°C (ThermoFisher, #EW-17705-10). After removing the supernatant, 250 μl of PBS 2% FBS was added to each well. The cells were mixed, and the plate was centrifuged again at 500 g for 3 minutes at 4°C (ThermoFisher, #EW-17705-10). After removing the supernatant, 100 μl of PBS 2% FBS antibody solution was added to each well. Antibody staining was performed on ice for 30 minutes. 150 μl of PBS 2% FBS was added to each well to wash out the cells from the unbound antibody. The plate was centrifuged at 500 g for 3 minutes at 4°C (ThermoFisher, #EW-17705-10), and the supernatant was removed. Finally, 200 μl of PBS (2% FBS) was added to each well, and the solution was filtered into a Falcon FACS tube (Coming, #352235) for FACS analysis.

[0217] CD69 was detected using aquamarine anti-mouse CD69 (Biolegend #104524). We used aquamarine Armenian hamster IgG isotype Ctrl (Biolegend #400925) as an isotype control.

[0218] result CD69 surface expression generated from different CAR constructs was tested. Data are presented in... Figure 8 In the middle. The relative counts for this diagram and subsequent diagrams are calculated relative to the pattern.

[0219] Figure 8 This is a FACS analysis of a 58C cell population transduced with appropriate anti-small molecule BAT-CAR T cells. CD69 was quantified as a result of CAR T cell activation upon antigen exposure. CD69 expression was triggered only when murine hybridoma T cells were engineered to recognize small molecules with anti-FITC CAR T (SEQ ID NO: 10) or anti-anthraquinone-2-ester (anti-AQ, SEQ ID NO: Y). No activation was observed when native 58C cells or 58C cells transduced with empty pMIG vectors were exposed to the antigen. Neither anti-FITC CAR T cells nor anti-AQ CAR T cells were activated in the presence of CAR-engineered murine hybridoma T cells as BSA effectors. The target was the antibody homologous antigen coated on the wells of a 24-well plate.

[0220] Example 5: Generation of MMP2-sensitive polymers for antigen exposure Materials and methods The purpose of this experiment was to demonstrate that chemically synthesized peptides can be cleaved by their homologous matrix metalloproteinases (MMPs) such as MMP2 or MMP9. The peptide can be cleaved into a free state in a solvent or directly or indirectly coupled to the tumor-targeting unit. The peptide was designed to couple to the antigen molecule and is intended to be a bridging block between the targeting unit and the masking PEG polymer.

[0221] Definition of masking peptides coupled with anthraquinone small molecules The selected peptide “CP(AQ)” (SEQ ID NO: 2) contains the MMP2 cleavage sequence at its center as revealed in Bremer et al., Nat Med. Jun, 7(6): 743-8 (2001). ProLeuGlyValArgGly "and the cleavage site is located between Gly and Val."

[0222] The CP(AQ) synthesis sequence is "Lys(AQ)SerGly ProLeuGlyValArgGly "SerSerCys": The first residue "Lys" is covalently bonded to the anthraquinone-2-acid of the antigen molecule via its γ-amino group. "SerGly" and "SerSer" surround its N-terminus and C-terminus, respectively, thereby providing the peptide with accessibility, flexibility, and increased aqueous solubility to the protease. Ultimately, the anthraquinone-2-acid is coupled to a lysine residue at the N-terminus of the peptide. To provide specific orientation during coupling of the peptide with the remaining platform components (e.g., tumor-targeting units and PEG-masking polymers), the conventional free amino group at the N-terminus of the cleavable peptide is retained, but the amino group at its C-terminus is added to L-cysteine. This amine is used for coupling to NHS-activated esters and L-cysteine ​​for direct coupling against maleimide.

[0223] Confirmation of peptide cleavage via MMP2 Thin-layer chromatography (TLC) was performed on UV fluorescent foil (Sigma-Aldrich, #70643) to separate and track the time-dependent peptide degradation caused by commercial MMP2 (BioLegend, #554304). Figure 9 ).

[0224] To track the protein degradation of CP(AQ) via MMP2, we obtained equal-volume samples of the cleaved peptide "Lys(AQ)SerGly". ProLeuGly "(pCP(AQ), SEQ ID NO: 1) is used as a reference during TLC operation.

[0225] It was found that a mobile phase consisting of CH₂Cl₂:MeOH = 3:1 (Sigma-Aldrich) was suitable for separating pCP(AQ) and CP(AQ). TLC was found to be suitable for this experiment because both pCP(AQ) and CP(AQ) are covalently bonded to anthraquinone-2-acid, which absorbs light in the UV range, thus quenching the fluorescence of the plate. Furthermore, CP(AQ) carries a positively charged arginine residue, significantly increasing the fragment polarity compared to the cleaved peptide pCP(AQ).

[0226] The protein degradation reaction was completed at 37°C with gentle shaking in 50 mM Borate buffer (pH 7.5) and 5 mM CaCl2 (Seltzer et al. J Biol Chem Nov 25;265(33):20409-13, (1990). 100 μg of C(AQ) and 0.2 μg of purified recombinant MMP2 in 100 μl of reaction buffer were added to this buffer. At regular time points, 1.5 μl of the reaction echelon was loaded onto the starting line. Two additional solutions were prepared with the reaction sample, one containing 100 μg of CP(AQ) in 100 μl of reaction buffer and the other containing 100 μg of pCP(AQ) in 100 μl of reaction buffer. Both solutions served as references during the separation process of the reaction sample and as degradation controls. The reaction was monitored under UV light in a conventional laboratory clean bench. (Apple iPhone) Take pictures of the board before and after it is in operation (5S).

[0227] result The results of CP(AQ) protein lysis via MMP2 showed that Figure 9 In comparison of spot intensities before and after TLC running, the results show that incubating CP(AQ) with MMP2 can transform the organic solution from hydrophilic to hydrophobic. Figure 9 (spot R). pCP(AQ) and CP(AQ) exhibit consistent and opposite behaviors, respectively. Figure 9 (spots F and P).

[0228] Example 6: Tumor antigen targeting via the BAT-CAR platform Materials and methods The purpose of this experiment was to demonstrate that the BAT-CAR platform can target tumor antigens by using its targeting unit alone or in association with its antigen-bearing domain. The BAT-CAR platform is modular and can be divided into two main structural domains: the tumor-targeting unit (TTU) and the antigen-bearing unit (CU). The platform's structural components are presented as follows: Figure 10 middle.

[0229] The TTU is sequentially divided into two domains: a tumor-targeting peptide and a TTU-CU linker region. The tumor-targeting peptide can be composed of an antibody against a tumor antigen, but can also be any peptide capable of recognizing proteins overexpressed on benign tumor cells with sufficient specificity. If the targeting unit is an antibody, it can be a full-length IgG antibody, a single-chain antibody (scFv), or a proteolytic product of the antigen-binding fragment (Fab) of native IgG. In this embodiment, the tumor-targeting peptide is the antibody trastuzumab (Genentech Inc., Herceptin). The TTU-CU linker region is composed of a bivalent antibody that recognizes a constant region on one side of the trastuzumab and a specific epitope on the CU on the other side. In this experiment, the TTU was incubated with Alexa fluor 647 (ThermoFisher Scientific, #A20347) to dissolve it for TTU tracking via flow cytometry (AMH).

[0230] The CU can be divided into four regions: peptide backbone, short PEG spacer region, protease-cleavable peptide (CP(AQ)) and PEG-masked tail.

[0231] The peptide backbone may consist of polylysine or polyglutamate or any polymer that allows side-chain coupling. It contains a region specifically recognized by the TTU-CU linker domain on the TTU and may be fluorescently labeled at one end. In this embodiment, the peptide backbone consists of polylysine and is FITC-labeled. The short PEG spacer region (Quanta Biodesign, PlainCity, OH 43064) may be a PEG linker group located between the peptide backbone and the CP(AQ). Following the short PEG linker group may be a different protease-cleavable peptide carrying the antigen molecule. In this embodiment, the protease-cleavable peptide is CP(AQ) (SEQ ID NO: 1). For comparison, in this embodiment, pCP(AQ) is also coupled to the short PEG linker group. In this embodiment, CP(AQ) may also be linked to Alexa fluor 647 (ThermoFisher Scientific, #A20347).

[0232] The PEG masking tail (Quanta Biodesign) can have varying lengths and complexities and can be terminally coupled to a fluorophore that is different from the fluorophore located on the peptide backbone after the masking PEG tail is released by the protease. In this embodiment, PEG12 and PEG24 terminally coupled to Alexa fluor 647 (ThermoFisher Scientific, #A20347) can be coupled to CP(AQ).

[0233] Quantification of tumor target binding via TTU To quantify the ability of TTU to target its homologous tumor antigens, human HER2 was introduced. + The cell line (HCC1954) was mixed with TTU / AMH or AMH alone. Furthermore, mouse skin melanoma HER2 was hatched with TTU / AMH. + Cell lines were used to provide a negative control. In this example, Herceptin-based anti-HER2-TTU / AMH was used.

[0234] To test the specificity of this TTU, 100,000 HER2 cells were harvested using a cell scraper (MedSupply Partners #TL-TR9000). + HCC1954 or HER2 - B16F10 cells. When necessary, these cells were placed on ice in 100 μl of PBS (2% FBS) with 1 μg of anti-HER2 inhibitor. - Incubate with TTU / AMH or 1 μg AMH for 30 minutes. After incubation, dilute with 400 μl of PBS 2% FBS and centrifuge at 500 g for 5 minutes at 4°C. Discard the supernatant and resuspend the cells in 200 μl of PBS 2% FBS. Finally, filter the cells through the cap of a Falcon FACS tube (Corning, #352235) and store the cells in a sealed Styrofoam incubator on ice for analysis.

[0235] Validation of target binding via TTU / CU To test anti-HER2-TTU / CU binding, 100,000 HER2 cells were harvested using a cell scraper (MedSupply Partners #TL-TR9000). + HCC1954 cells. Cells were stained with 0.4 μg of a TTU module and 0.4 μg of a CU module with a different architecture, wherein the TTU module was the same as previously described but not linked to any fluorophore. Figure 10All CUs used here have been labeled with FITC located in their framework. The second sample was also labeled with -PEG2-pCP(AQ), the third sample with -PEG2-CP(AQ)-A647, and the fourth sample with -PEG2-CP(AQ)-PEG. 12 -Alexa fluor 647 labeled, the last sample was labeled -PEG2-CP(AQ)-PEG 24 - Alexa fluor 647 label. Add both TTU and CU simultaneously to the cells. Stain the cells on ice for 30 minutes, or at 37°C and 5% CO2 for 8 hours.

[0236] After incubation at 37°C, cells were scraped off and the supernatant (500 μl) was transferred to a microcentrifuge tube. The cells were then diluted with 500 μl of PBS (2% FBS) and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded, and the solids were washed with 1000 μl of PBS (2% FBS). Finally, the cells were resuspended in 200 μl of PBS (2% FBS) and filtered through the cap of a FalconFACS tube (Corning, #352235). The cells were then kept in a sealed Styrofoam incubator on ice until analysis.

[0237] result Target specificity obtained via TTU / AMH was shown to be... Figure 11 In the middle. Unstained HER2 + Histogram of HCC1954 cells and HER2 - Histogram overlap of B16F10 cells indicates anti-HER2 - TTU / AMH does not target sites outside the target site. As expected, when using this anti-HER2... - TTU / AMH complex incubates HER2 + When HCC1954 cells were used, a strong Alexafluor 647 signal was observed.

[0238] Data collected via FACS were used to verify the different mixtures passing through TTU / CU. Figure 10 Examples of tumor target binding data obtained from the construction of E) are summarized in Figure 12 middle.

[0239] Figure 12 It is anti-HER2 - TTU and variable CU to HER2 + HCC1954 cells were stained. The image above shows the staining with CU-FITC-CP(AQ)-PEG. 24Data from HCC1954 cells stained with Alexa fluor 647 ± TTU. The acquired data showed that the addition of TTU enhanced the binding of CU to the target tumor cells. The figure below shows the binding of CU-FITC-CP(AQ)-PEG. 24 Data from intact cell populations stained with Alexa fluor 647 + TTU after 30 minutes of incubation on ice and 8 hours of incubation at 37°C / 5% CO2. The data collected confirm minimal CP(AQ) lysis during the 8-hour incubation period, as cells with the same FITC intensity showed a lower intensity of A646 signal. Figure 7 In the image above, the target is HCC1954 cells, and the effectors are ±TTU and CU-FITC-CP(AQ)-PEG. 24 -Alexa fluor 647.

[0240] Example 7: Construction of anti-FITC CAR T and its components Structure #1 encodes a polypeptide containing "anti-FITC CAR T". Anti-FITC-4M5.3 antibody Signal peptide – CD8a-sp|P01731|1-27 Hinge area – CD8a-sp|P01731|152-194 Transmembrane region (TM) –CD28-sp|P31041|151-177 Intracellular domain (ICD) –CD28-sp|P31041|178-218 Intracellular domain (ICD) –41BB-sp|P20334|209-256 Intracellular domain (ICD) –CD3z-sp|P241614|52-164 Anti-FITC CAR T total peptide Other specific embodiments Although the invention has been disclosed together with its detailed description, the foregoing description is intended to be illustrative rather than limiting of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims

1. A system comprising: A binding molecule with tumor antigen specificity, among which, The binding molecule comprises a small molecule covalently linked to a targeting moiety, wherein the targeting moiety specifically binds to the tumor antigen; and A cell containing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain, the extracellular domain specifically binding small molecules of the binding molecule.

2. The system as claimed in claim 1, wherein, The target component is an antibody, affinity, aptamer, or T-cell receptor multimer.

3. The system as described in claim 2, wherein, The antibody is either Fab or scFV.

4. The system as described in claim 2, wherein, The T-cell receptor multimer is a tetramer.

5. The system as described in any one of claims 1 to 4, wherein, The size of this small molecule is less than 5 kDa.

6. The system as described in any one of claims 1 to 4, wherein, This small molecule is a synthetic inorganic or organic compound.

7. The system as described in any one of claims 1 to 4, wherein, This extracellular domain is an antibody.

8. The system of claim 7, wherein, The antibody is either Fab or scFV.

9. The system as claimed in any one of claims 1 to 4, wherein, The transmembrane domain also includes a stalk region located between the extracellular domain and the transmembrane domain.

10. The system as claimed in any one of claims 1 to 4, wherein, This transmembrane domain contains CD28.

11. The system of any one of claims 1 to 4, further comprising one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signal transduction domain.

12. The system of claim 11, wherein, The one or more additional co-stimulatory molecules include CD3ζ, CD28, 4-1BB, 4-1BBL, ICOS, or OX40.

13. The system as claimed in any one of claims 1 to 4, wherein, This intracellular signal transduction domain contains the CD3ζ chain.

14. The system as claimed in any one of claims 1 to 4, wherein, This cell is a T cell.

15. The system of claim 14, wherein, This T cell is a CD4 cell. + T cells and / or CD8 + T cells.

16. The system of claim 14, wherein, This T cell is a regulatory T cell or a follicle-regulating T cell.

17. The system as claimed in any one of claims 1 to 4, wherein, The small molecule is fluorescein isothiocyanate.

18. The system of claim 17, wherein, The extracellular domain includes the anti-FITC-4M5.3 antibody of SEQ ID NO:

3.

19. The system as claimed in any one of claims 1 to 4, wherein, The small molecule is linked to a protective domain to mask it.

20. The system of claim 19, wherein, The protected area includes: Linked to the carrier domain of the protease-susceptible peptide; Masking peptides and masking polymers; or pH-sensitive protective domain.

21. Use of the system of any one of claims 1 to 20 in the preparation of a medicament for treating cancer in a subject in need.

22. The use as described in claim 21, wherein, The subject was given the binding molecule in the first period and the cell in the second period.

23. A chimeric antigen receptor comprising an extracellular domain of an intracellular signal transduction domain, a transmembrane domain, and a binding recognition domain.

24. A genetically engineered cell that expresses and loads a chimeric antigen receptor on its cell surface membrane, wherein, This chimeric antigen receptor comprises an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain that binds to and recognizes the antigen.

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