Compositions and methods for treating cancer with DuoCAR

Abstract

To provide a novel immunotherapeutic composition based on a chimeric antigen receptor (CAR)-T cell useful in the treatment of cancer (e.g., leukemia, lymphoma, multiple myeloma), inflammatory diseases, autoimmune diseases, and the like, and to provide uses thereof.SOLUTION: Provided is an immunotherapeutic composition comprising at least one polycistronic vector comprising a promoter operably linked to a polycistronic nucleic acid sequence. The polycistronic nucleic acid sequence encodes two or more functional CARs comprising: an extracellular antigen binding domain; a transmembrane domain; and one or more non-identical intracellular signaling motifs each comprising a non-identical amino acid sequence independently selected from a group consisting of particular amino acid sequences. The at least one polycistronic vector is used to genetically modify one or more lymphocyte populations.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference with related applications This PCT patent application claims priority based on U.S. Utility Patent Application No. 16 / 078,269, filed on 21 August 2018, which is a continuation-in-part application of U.S. Utility Patent Application No. 16 / 134,735, filed on 1 September 2018, which claims priority based on U.S. Provisional Patent Application No. 62 / 382,791, filed on 2 September 2016 in accordance with Section 119(e) of the U.S. Patent Act, and the entire contents of each of these are incorporated herein by reference.

[0002] Areas of disclosure This application relates to the field of cancer, and more specifically to a composition comprising at least two vectors encoding a functional chimeric antigen receptor, and to a method of using the same in patient-specific immunotherapy.

[0003] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The filename of this ASCII copy (created on September 16, 2019) is "Sequence Listing.txt" and its size is 236 kilobytes. [Background technology]

[0004] Background of the Invention Cancer is one of the most deadly threats to human health. Nearly 1.3 million people are diagnosed with cancer each year in the United States alone, making it the second leading cause of death after cardiovascular disease, and accounting for approximately one in four deaths. Most of these deaths are due to solid tumors. While medical treatments for some specific cancers have advanced significantly, the five-year survival rate for all cancers combined has improved by only about 10% in the last 20 years. Cancer or malignant tumors are extremely difficult to treat because their metastasis and growth are rapid and uncontrollable. One of the challenges in modern cancer treatment is ensuring that patients receive effective treatment after biopsy and cancer diagnosis. This refers to the time elapsed before treatment is received. During this time, the patient's tumor can grow unimpeded, resulting in further disease progression before treatment can be performed. This negatively impacts the prognosis and outcome of the cancer.

[0005] Chimeric antigen receptors (DuoCARs) are hybrid components consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a binding / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2 (4): e23621). The antigen-binding motif of a CAR is generally immunoglobulin (Ig) It is constructed by mimicking the single-chain fragment variable (ScFv), which is the smallest binding domain of the molecule. Other antigen-binding motifs such as receptor ligands (i.e., IL-13 designed to bind to the IL-13 receptor expressed in tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) are also incorporated by design. Other cell targets that express CARs (such as NK or gamma-delta T cells) are also being constructed (Brown CE et al Clin Cancer). Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012; 7 (2): e31210). This study aimed to identify the most active T cell population transduced using CAR vectors, determine optimal culture and proliferation techniques, and elucidate the molecular structure of the CAR protein itself in detail. Further considerable effort will be required to clarify this.

[0006] The binding motif of a CAR may be a relatively stable structural domain, such as the constant domain of IgG, or it can be designed as a long, mobile linker. Using structural motifs such as those derived from the IgG constant domain, the scFv binding domain can be extended to locations far from the T cell membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., the disialoganglioside GD2; Orentas et al., this observation unpublished). All signaling motifs used in CARs to date have included a CD3-zeta chain, as this core motif is a crucial signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and a CD28 transmembrane sequence. This motif was used in third-generation CARs further containing the CD137(4-1BB) signaling motif (Zhao Y et al J Immunol. 2009; 183 (9): 5563-74). With the emergence of new technologies, the activation of T cells by beads bound to anti-CD3 and anti-CD28 antibodies, as well as the existence of the classical "signal 2" from CD28, no longer need to be encoded by the CAR itself. Third-generation vectors using bead activation have been found not to outperform second-generation vectors in in vitro assays, and furthermore, they did not provide any significant advantages over second-generation vectors in leukemia mouse models (Haso W, Lee DW, Shah NN, Stetler-Stevenson). M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7):1165-74; Kochenderfer JN et al. Blood. 2012; 119 (12):2709-20). This relates to the second generation CD28 / CD3-zeta (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-zeta signaling pathways. This has been demonstrated by the clinical success of CD19-specific CARs in the T-cell model (Porter DL et al. N Engl J Med. 2011; 365 (8): 725-33). In addition to CD137, other members of the tumor necrosis factor receptor superfamily, such as OX40, can also provide important sustained signaling in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T cell population is cultured.

[0007] At present, a challenge in more broadly and effectively applying CAR therapy to cancer is the lack of promising targets. While the creation of binders that bind to cell surface antigens is now easily achievable, the discovery of cell surface antigens that are tumor-specific and do not affect normal tissue remains extremely difficult. One method that can give CAR-expressing T cells stronger target cell specificity than before is to use a combination of multiple CAR approaches. In one system, the CD3-zeta signaling unit and CD28 signaling unit are separated into two different CAR constructs expressed in the same cell, and in another system, the two DuoCARs are expressed in the same T cell, however, since one of them has lower affinity, the other CAR must be bound first to maximize the activity of the latter CAR (Lanitis E et al. Cancer Immunol Res. 2013;1(1):43-53; Kloss CC et al. Nat Biotechnol. 2013;31(1):71-5). A second challenge in developing scFv-based CARs as immunotherapeutic agents is the non-uniformity of tumor cells. At least one group has developed a CAR-based treatment for glioblastoma, in which the effector cell population simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2), attempting to avoid proliferation of populations lacking the target antigens (Hegde M et al. Mol Ther. 2013;21(11):2087-101). .

[0008] T cell-based immunotherapy has become a cutting-edge field in synthetic biology. Multiple promoters and gene products have been conceived to induce these highly potent cells into the tumor microenvironment, where T cells can circumvent negative regulatory signals and mediate effective tumor death. Drug-induced dimerization of the inducible caspase 9 construct using AP1903 to remove unwanted T cells presents one strategy for pharmacologically activating a powerful switch that can control the T cell population (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Furthermore, by expressing a decoy receptor, an effector T cell population that does not respond to the negative regulatory effects of transforming growth factor β has been created, demonstrating the extent to which effector T cells can be manipulated to achieve optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5).

[0009] Therefore, although CARs can trigger T cell activation in a manner similar to endogenous T cell receptors, the clinical application of CAR-based technologies has not progressed at present due to major obstacles such as the limited proliferation of CAR+ T cells in vivo, the rapid disappearance of these cells after infusion, which hinders the realization of clinical activity, the recurrence of the underlying medical disease or condition, and the excessive time it takes to treat cancer in a timely manner using such CAR+ T cells after diagnosis. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Therefore, there has been an urgent and long-standing need in the art to discover compositions and methods for treating cancer by using CAR-based therapies that can exhibit cancer-specific and intended therapeutic properties without presenting the aforementioned problems. [Means for solving the problem]

[0011] The present invention addresses the above-described need by providing a composition comprising at least two vectors encoding a functional chimeric antigen receptor, and methods of using the same in patient-specific immunotherapy for treating cancer and other diseases and / or conditions.

[0012] In particular, the present invention, as disclosed and described herein, provides an immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least two vectors, each vector encoding a functional DuoCAR, such that upon combination of the vectors, expression of two or more non-identical binding domains results, and the binding domains encoded by the vectors are each covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and the immunotherapeutic composition can be used to transduce autologous lymphocytes to generate an active patient-specific anti-tumor lymphocyte cell population, which can be returned to the patient by direct injection to promote in vivo proliferation and persistence of patient-specific anti-tumor T cells, and as a result, can patient-specifically result in tumor stabilization, shrinkage, elimination, cancer remission, or prevention or remission of cancer recurrence, or combinations thereof.

[0013] SUMMARY OF THE INVENTION A novel adoptive immunotherapeutic composition comprising lymphocytes transduced with two or more vectors, and methods of using the same in patient-specific combination immunotherapy for treating cancer and other diseases and conditions are provided herein.

[0014] Thus, in one aspect, a lentiviral vector expressing a Duo chimeric antigen receptor (DuoCAR), and a nucleic acid molecule encoding the lentiviral vector expressing DuoCAR are provided herein. Also provided are methods of using the disclosed lentiviral vector, host cell, and nucleic acid molecule for treating, for example, cancer in a subject.

[0015] ​ In one aspect, there is provided an immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each vector encoding a functional CAR, and at least one binding domain in one of the vectors not being identical, such that as a result of the combination of the vectors, the expression of two or more non-identical binding domains occurs, and the binding domains encoded by the vectors are each covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0016] In one embodiment, there is provided an immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least three vectors (TrioCARs), each vector encoding a functional CAR, such that as a result of the combination of the vectors, the expression of two or more non-identical binding domains occurs, and the binding domains encoded by the vectors are each covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0017] In one embodiment, there is provided an immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least four vectors (QuatroCARs), each vector encoding a functional CAR, such that as a result of the combination of the vectors, the expression of two or more non-identical binding domains occurs, and the binding domains encoded by the vectors are each covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0018] In yet another embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 vectors (e.g., “nCAR”), each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains as a result of the combination of vectors, each of which binding domains encoded by the vectors is covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs, and each of the unique components of the nCAR set is incorporated into a CAR product to constitute a unique CAR composition referred to herein as “nCAR” (e.g., DuoCAR, TrioCAR, QuatroCAR, PentaCAR, HexaCAR, HeptaCAR, OctaCAR, NonaCAR, and DecaCAR, etc.).

[0019] In one embodiment, an immunotherapy composition is provided, which comprises (a) at least two vectors, each containing a nucleic acid sequence capable of functioning in a cell; (b) each vector encoding a functional CAR; (c) each CAR comprising at least one binding domain, one transmembrane domain, and at least one intracellular signaling motif; (d) at least one binding domain in one of the vectors is not identical; and (e) at least one binding domain, one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of the vectors is used to genetically modify one or more lymphocyte populations.

[0020] In another embodiment, an immunotherapy composition is provided, which comprises (a) at least two vectors each comprising a nucleic acid sequence capable of functioning in a cell, (b) each vector encoding a functional CAR, (c) each CAR comprising at least one binding domain, one transmembrane domain, and at least one intracellular signaling motif, (d) the at least one binding domain in each vector is not identical, and (e) the few (f) at least one combination of signaling motifs is not identical between each of the vectors, and at least one binding domain, one transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0021] In one embodiment, an immunotherapeutic composition is provided, in which each vector encodes one or more functional CARs.

[0022] In another embodiment, an immunotherapeutic composition is provided, in which a combination of one or more signaling motifs is identical on one or more vectors.

[0023] In another embodiment, an immunotherapeutic composition is provided, in which one or more binding domains are identical on one or more vectors.

[0024] In another embodiment, an immunotherapeutic composition is provided, in which the lymphocyte population comprises autologous T cells or a mixture of peripheral blood-derived lymphocytes.

[0025] In another embodiment, an immunotherapeutic composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one single-strand variable fragment of an antibody that binds to the antigen.

[0026] In another embodiment, an immunotherapeutic composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one heavy chain variable region of an antibody that binds to the antigen.

[0027] In another embodiment, an immunotherapeutic composition is provided, in which at least one extracellular antigen-binding domain of CAR, at least one intracellular signaling domain of CAR, or both, is bound to a transmembrane domain by a linker domain or a spacer domain.

[0028] In another embodiment, an immunotherapeutic composition is provided, in which the extracellular antigen-binding domain of the CAR is located behind the leader peptide.

[0029] In another embodiment, an immunotherapeutic composition is provided in which the extracellular antigen-binding domain of CAR targets antigens including CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1, MAGE-A3, PRAME peptide and MHC combinations, or any combination thereof.

[0030] In another embodiment, an immunotherapeutic composition is provided, in which the extracellular antigen-binding domains of CAR are anti-CD19 scFV antigen-binding domains, anti-CD20 scFV antigen-binding domains, anti-CD22 scFV antigen-binding domains, anti-ROR1 scFV antigen-binding domains, anti-TSLPR scFV antigen-binding domains, anti-mesoterin scFV antigen-binding domains, anti-CD33 scFV antigen-binding domains, anti-CD38 scFV antigen-binding domains, anti-CD123 (IL3RA) scFV antigen-binding domains, anti-CD138 scFV antigen-binding domains, anti-BCMA (CD269) scFV antigen-binding domains, anti-GPC2 scFV antigen-binding domains, anti-GPC3 scFV antigen-binding domains, anti-FGFR4 scFV antigen-binding domains, anti-c-Met scFV antigen-binding domains, and anti-PMSA scFV Antigen-binding domain, anti-glycolipid F77 scFV antigen-binding domain, anti-EGFRvIII scFV antigen-binding domain, anti-GD-2 scFV antigen-binding domain, anti-NY-ESO-1 TCR (including single-stranded TCR construct) antigen-binding domain, anti-MAGE-A3 This includes TCRs, or amino acid sequences that are 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them, or any combination thereof.

[0031] In another embodiment, an immunotherapeutic composition is provided in which the linker domain or spacer domain of CAR is derived from the extracellular domain of CD8 and bound to the transmembrane domain.

[0032] In another embodiment, an immunotherapeutic composition is provided, wherein CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, Fc epsilon R, or any combination thereof.

[0033] In another embodiment, an immunotherapeutic composition is provided, wherein at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0034] In another embodiment, an immunotherapeutic composition is provided, in which at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

[0035] In another embodiment, an immunotherapeutic composition is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0036] In another embodiment, an immunotherapeutic composition is provided, wherein at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), PD-1, GITR, CTLA-4, or any combination thereof.

[0037] In another embodiment, an immunotherapeutic composition is provided, in which one vector is used to encode any chimeric antigen receptor, combined with a CRISPR system for incorporation (e.g., retrovirus, adenovirus, SV40, herpes vector, POX vector, RNA, plasmid, cosmid, or any viral or non-viral vector).

[0038] In another embodiment, an immunotherapy composition in which each vector is an RNA vector or a DNA vector is provided alone or in combination with a transfection reagent or a method for delivering RNA or DNA into cells (including, but not limited to, electroporation).

[0039] In another embodiment, an immunotherapeutic composition is provided, in which at least one vector expresses a nucleic acid molecule that modulates the expression of nucleic acids in a cell.

[0040] In another embodiment, an immunotherapeutic composition is provided in which nucleic acid molecules inhibit or cause loss of expression of endogenous genes.

[0041] In certain embodiments, an immunotherapeutic composition is provided, in which an active patient-specific autologous antitumor lymphocyte population is generated within 1, 2, 3, 4, 5, 7, 10, 12, 14, 21 days, or 1 month from lymphocyte retrieval or tumor biopsy, and by returning the active patient-specific autologous antitumor lymphocyte population to the cancer patient by infusion, the in vivo proliferation and persistence of the patient-specific antitumor lymphocytes can be promoted, and as a result, tumor stabilization, reduction, removal, cancer remission, or prevention or remission of cancer recurrence, or a combination thereof, can be brought about in a patient-specific manner.

[0042] In one embodiment, isolated nucleic acid molecules encoding the above-described chimeric antigen receptor are provided herein.

[0043] In one embodiment of DuoCAR used in a patient-specific autologous lymphocyte population of an immunotherapy composition according to the present invention, DuoCAR is modified to express or contain a detectable marker for use in the diagnosis, monitoring, and / or prediction of treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progress of such treatment. In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, the nucleic acid molecule encoding the disclosed DuoCAR may be contained in a vector such as a viral vector or a non-viral vector. The vector may be a DNA vector, RNA vector, plasmid vector, cosmid vector, herpesvirus vector, measles virus vector, lentivirus vector, adenovirus vector, or retrovirus vector, or a combination thereof.

[0044] In certain embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, two or more lentiviral vectors include, for example, retroviral GPs such as bitrophic mouse leukemia virus [MLV-A], baboon endogenous virus (BaEV), GP164, gibbon leukemia virus [GALV], RD114, and feline endogenous virus, as well as varicella stomatitis virus [VSV], measles virus, avian plague virus [FPV], Ebola virus [Ebo V], and lymphocytic choriomeningitis virus [LCMV]. They are pseudotyped with different viral glycoproteins (GPs), including, but not limited to, non-retroviral GPs such as ] and their chimeric variants (e.g., chimeric GPs encoding the extracellular and transmembrane domains of GALV, or RD114 GP fused to the cytoplasmic end (designated TR) of MLV-A GP).

[0045] In certain embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, the vector further comprises a promoter, which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0046] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, the vector expressing the CAR may be further modified to include one or more activatable elements for controlling the expression of CAR T cells or for eliminating CAR-T cells by a suicide switch. This suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0047] In another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, host cells containing the nucleic acid molecule encoding DuoCAR are further provided. In several embodiments, the host cells are T cells, such as primary T cells obtained from a subject. In one embodiment, the host cells are CD8+ T cells. In another embodiment, the host cells are CD4+ T cells. In one embodiment, the host cells are selected without regard to balance from CD4+ lymphocytes and CD8+ lymphocytes purified directly from the patient product. In yet another embodiment, the number of CD4+ T cells and CD8+ T cells in the product is a specific number. In yet another embodiment, a specific T cell subset is identified and utilized by phenotypic markers including T naive cells (Tn), T effector memory cells (Tem), T central memory cells (Tcm), T regulatory cells (Treg), inducible T regulatory cells (iTreg), T suppressor cells (Ts), T stem cell memory cells (Tscm), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.

[0048] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of an immunotherapy composition comprising a population of patient-specific autologous antitumor lymphocyte cells of a human patient with cancer, wherein the cells of the population comprise cells containing nucleic acid molecules encoding at least two vectors, each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains as a result of the vectors being combined, each of the binding domains encoded by the vectors being covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0049] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of an immunotherapy composition comprising a population of patient-specific autologous antitumor lymphocyte cells of a human patient with cancer, wherein the cells of the population comprise (a) cells comprising nucleic acid molecules encoding two or more vectors, (b) each vector encoding a functional CAR, (c) each CAR comprising at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif, (d) at least one binding domain in one of the vectors is not identical, and (e) at least one binding domain, one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of the vectors is used to genetically modify one or more lymphocyte populations.

[0050] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of an immunotherapy composition comprising a population of patient-specific autologous antitumor lymphocyte cells of a human patient with cancer, wherein the cells of the population comprise (a) cells comprising nucleic acid molecules encoding two or more vectors, (b) each vector encoding a functional CAR, (c) each CAR comprising at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif, (d) at least one binding domain in each vector is not identical, (e) the combination of the at least one signaling motif is not identical among the respective vectors, and (f) the at least one binding domain, one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of the two or more vectors is used to genetically modify one or more lymphocyte populations.

[0051] In one embodiment, the cancer is a refractory cancer that does not respond to one or more chemotherapy agents. The cancer includes hematopoietic cancers, myelodysplastic syndromes, pancreatic cancer, head and neck cancers, skin tumors, acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood cancers and solid tumors, or any combination thereof. In another embodiment, This includes blood cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma, or any combination thereof.

[0052] In yet another embodiment, cancer includes oral (coral) and pharyngeal cancers (tongue, mouth, pharynx, head and neck), gastrointestinal cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory cancers (larynx, lung, and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), adult cancers, pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovaries, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eye and orbit, endocrine system (thyroid), brain and other nervous system, or any combination thereof.

[0053] In another embodiment, a pharmaceutical composition is provided comprising a population of autologous lymphocyte cells transduced with two or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs), thereby promoting the in vivo proliferation and persistence of patient-specific antitumor T cells, and consequently generating a patient-specific population of autologous antitumor lymphocyte cells that can result in tumor stabilization, reduction, removal, cancer remission, or prevention or remission of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0054] In another embodiment, a pharmaceutical composition is provided comprising an autologous T cell population transduced with one or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs), thereby promoting the in vivo proliferation and persistence of patient-specific antitumor T cells, resulting in a patient-specific autologous antitumor lymphocyte population that can lead to tumor stabilization, reduction, removal, cancer remission, or prevention or remission of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0055] In another embodiment, a method for producing active patient-specific autologous antitumor DuoCAR-containing lymphocytes is provided. This method includes the step of transducing lymphocytes with two or more vectors or nucleic acid molecules encoding two or more chimeric antigen receptors (DuoCARs) that specifically bind to an antigen, thereby producing active patient-specific autologous antitumor DuoCAR-containing lymphocytes.

[0056] In yet another embodiment, a method is provided for generating a population of RNA-modified lymphocyte cells, which includes the step of generating a patient-specific autologous antitumor lymphocyte cell population, which can thereby promote the in vivo proliferation and persistence of patient-specific antitumor T cells, and consequently result in patient-specific stabilization, reduction, or removal of tumors, remission of cancer, or prevention or remission of cancer recurrence, or a combination thereof.

[0057] In another embodiment, a method is provided for treating mammals having a disease, disorder, or condition associated with high expression of tumor antigens, the method comprising administering to a subject a pharmaceutical composition containing an antitumor-effective amount of an autologous lymphocyte cell population transduced with one or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs), thereby promoting the in vivo proliferation and persistence of patient-specific antitumor T cells, and consequently resulting in patient-specific stabilization, reduction, or removal of tumors, remission of cancer, or prevention or remission of cancer recurrence, or a combination thereof, patient-specific autologous antitumor The process includes generating a population of ulcerative lymphocyte cells.

[0058] In another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with high expression of a tumor antigen, the method comprising administering to a subject a pharmaceutical composition containing an antitumor-effective amount of an autologous lymphocyte cell population transduced with two or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs) to generate a patient-specific autologous antitumor lymphocyte cell population, which can be returned to the patient by direct injection to promote in vivo proliferation and persistence of patient-specific antitumor T cells, and as a result can be patient-specifically brought about tumor stabilization, reduction, removal, or remission of cancer, or prevention or remission of cancer recurrence, or any combination thereof.

[0059] In one embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with high expression of a tumor antigen, the method comprising the step of administering a pharmaceutical composition comprising at least two vectors, each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains as a result of the combination of vectors, each binding domain encoded by the vectors being covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs, the pharmaceutical composition also comprising pharmaceutically acceptable excipients, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0060] In another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with high expression of a tumor antigen, the method comprising the step of administering a pharmaceutical composition to a subject, wherein (a) the pharmaceutical composition comprises nucleic acid molecules encoding two or more vectors, (b) each vector encoding a functional CAR, (c) each CAR comprising at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif, (d) at least one binding domain in one of the vectors is not identical, and (e) at least one binding domain, one transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, the combination of vectors being used to genetically modify one or more lymphocyte populations.

[0061] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with high expression of a tumor antigen, the method comprising the step of administering a pharmaceutical composition to a subject, wherein (a) the pharmaceutical composition comprises nucleic acid molecules encoding two or more vectors, (b) each vector encoding a functional CAR, (c) each CAR comprising at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif, (d) the at least one binding domain in each vector is not identical, (e) the combination of the at least one signaling motif is not identical among the respective vectors, and (f) the at least one binding domain, one transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of the two or more vectors is used to genetically modify one or more lymphocyte populations.

[0062] In certain embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, which are returned to the patient by direct infusion to prevent relapse or induce remission of a malignant disease.

[0063] In certain other embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, where autologous lymphocytes are patient-specific antitumor T-cell lymphocytes in vivo. It is returned to the patient by direct injection to promote growth and persistence, and as a result to bring about patient-specific outcomes such as tumor stabilization, reduction, removal, or remission of cancer, or prevention or remission of cancer recurrence, or any combination thereof.

[0064] In yet another embodiment, T cells are pre-selected based on specific activation or expression of memory-related surface markers.

[0065] In yet another embodiment, the T cells are derived from a hematopoietic stem cell donor, and the procedure described above is performed in the context of hematopoietic stem cell transplantation.

[0066] In certain embodiments, a method is provided in which lymphocytes are pre-selected based on specific activation or expression of memory-related surface markers.

[0067] In certain embodiments, a method is provided herein in which the lymphocyte cells are T cells derived from a hematopoietic stem cell donor and the procedure described above is performed in the context of hematopoietic stem cell transplantation.

[0068] In yet another embodiment, a method is provided for generating a persistent population of genetically modified patient-specific autologous antitumor lymphocyte cells in a person diagnosed with cancer. In one embodiment, the method comprises administering one or more patient-specific autologous antitumor lymphocyte cell populations described herein to a human patient in need thereof, wherein the persistent population of patient-specific autologous antitumor lymphocyte cells, or the population of offspring of such lymphocyte cells, persists in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years after administration.

[0069] In one embodiment, the progeny lymphocytes in humans include memory T cells. In another embodiment, the T cells are autologous T cells.

[0070] In all aspects and embodiments of the methods described herein, any cancer, disease, disorder, or condition associated with the high expression of tumor antigens described above may be treated, prevented, or remitted using a patient-specific autologous antitumor lymphocyte cell population comprising one or more of the Duo Car immunotherapy compositions disclosed herein.

[0071] In yet another embodiment, a kit is provided for preparing a DuoCar immunotherapy composition comprising a patient-specific autologous antitumor lymphocyte cell population, as described above, or for preventing, treating, or relieving any cancer, disease, disorder, or condition associated with high expression of tumor antigens in a subject, as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for use of the kit.

[0072] The compositions and methods according to the present invention have been illustrated above with reference to the fabrication and use of DuoCAR, but in this specification, it is particularly intended that the compositions and methods include the fabrication and use of TrioCAR and QuatroCAR.

[0073] In yet another embodiment, the immunotherapeutic composition comprises one or more isolated nucleic acid molecules encoding at least one vector, wherein the vector contains a nucleic acid sequence resulting in at least one messenger RNA encoding DuoCAR (i.e., a multi-cistronic nucleic acid, or a nucleic acid resulting in one or more transcripts), and as a result, the ability to bind to two or more non-identical antigen targets. This results in multiple antigen specificity in a single cell expressing the vector.

[0074] In yet another embodiment, the immunotherapeutic composition comprises one or more isolated nucleic acid molecules encoding at least two vectors, each vector further encoding a functional tag or anti-tag binding site (AT-CAR) that, when incubated or co-administered with a soluble binder (such as a tagged scFv or an scFv bound to an anti-tag binder), reconstitutes a functional chimeric antigen receptor, thereby resulting in the ability to bind to two or more non-identical antigen-binding domains as a result of the combination of these two vectors, thereby creating multi-antigen specificity in cells expressing these two vectors.

[0075] In yet another embodiment, the immunotherapeutic composition comprises one or more isolated nucleic acid molecules encoding at least two vectors, each vector encoding a functional tag or anti-tag binding site (AT-CAR) that, when incubated or co-administered with a soluble binder (such as a tagged scFv or an scFv bound to an anti-tag binder), reconstitutes a functional chimeric antigen receptor, wherein each vector expresses a unique tag (or anti-tag) that can bind to a soluble protein or protein modification structure to result in multi-antigen specificity, or each vector expresses a unique tag (or anti-tag) that binds to only one of the soluble binding domains to result in specific binding of an intracellular signaling motif encoded by the AT-CAR to the antigen-binding domain of the tagged (or anti-tagged) binder.

[0076] In the embodiments (without limitation) of each of the DuoCAR vector preparations, compositions, and methods disclosed in the embodiments and aspects described above, the two vectors can be prepared separately and then added to T cells sequentially or simultaneously. In another embodiment (without limitation), the plasmid DNA of two or more vectors can be conjugated before or during transfection of production cells, or integrated into the genome of production cells, to produce a mixture of viral vectors containing multiple DuoCAR vector particles, which can then be used for transduction and genetic modification of patient T cells.

[0077] For each of the various aspects and embodiments of DuoCAR, TrioCAR, and QuatroCAR as specifically intended herein, the nucleotide sequence encoding the functional CAR includes the nucleotide sequence of SEQ ID NOs: 3, 9, 21, 25, 29, 31, 35, 39, 43, 47, 49, 51, 53, 55, 59, or 61, or any combination thereof.

[0078] For each of the various forms and embodiments of DuoCAR, TrioCAR, and QuatroCAR as specifically intended herein, each vector encodes a functional CAR containing the amino acid sequence of SEQ ID NOs: 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62, or any combination thereof.

[0079] The patient-specific autologous antitumor lymphocyte cell populations, two or more lentiviral vectors expressing chimeric antigen receptors (DuoCAR), host cells, and methods described above are understood to be useful beyond the scope of the specific embodiments and models described in detail herein. The features and advantages of the above-described disclosure will become even clearer from the following detailed description with reference to the accompanying drawings.

[0080] Brief explanation of the drawing The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show currently preferred embodiments. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. [Brief explanation of the drawing]

[0081] [Figure 1] Four products (Examples 1-4) that can be prepared as separate commercial products are shown. These DuoCAR sets can be prepared to target human B-cell malignancies expressing three leukemia-associated antigens: CD19, CD20, and CD22. In Product 1, two gene vectors are used to simultaneously transduce an activated T cell population. The first vector encodes two antigen-binding domains (CD19, CD20) bound to one intracellular domain (z, CD3 zeta chain) connected by a CD8 transmembrane domain (8). The second vector encodes a CD22-binding domain and two signaling domains (BB, derived from CD137 / 4-1BB; and z). The second product (Example 2) has the first vector plus CD19 and CD20-binding domains bound to the CD28 and z signaling domains. The second vector encodes a CD22-binding domain and BB and z-signaling domains, essentially replicating the signaling package (three distinct signaling domains) of a third-generation CAR vector. In the third product (Example 3), the first vector encodes CD20 and CD22-binding domains bound to the BB and z-signaling domains, and the second vector encodes a CD19-binding domain bound to the CD28 and z-signaling domains. In the fourth product (Example 4), the first vector encodes CD20 and CD22-binding domains and BB and z-signaling domains. The second vector encodes a CD19-binding domain and a z-signaling domain. [Figure 2]This shows all the individual elements that can be incorporated into DuoCAR for therapeutic products targeting B-cell malignancies. Scientific terminology is the same as in Figure 1. [Figure 3] A general scheme for DuoCARs applicable to multiple therapeutic needs, including inflammatory or autoimmune diseases and infections, is presented. In the figure, a-CDX, a-CDY, and a-CDZ refer to antigen-binding domains specific to each of the three different target antigens CDX, CDY, and CDZ. The intracellular portion of each CAR contains a CD8 linker-transmembrane domain bound to either the CD3-zeta, CD28, or 4-1BB signaling domain (as shown in Figure 1). Specific combinations of any two of these vectors (e.g., A and F, where antigens X, Y, and Z are targeted and intracellular signaling is provided by CD3-zeta and 4-1BB) can be incorporated into a single vector, depending on the specific therapeutic need. [Figure 4] A general scheme for DuoCAR sets, where each vector targets two antigens, is shown. The same vectors in Figure 3 also have the same individual letter names (A is the same in both Figure 3 and Figure 4). A novel fourth antigen-binding domain is shown as a-CDW. One product that can target four antigens is the A+T DuoCAR set. In this case, the extracellular antigens CDX, CDY, CDZ, and CDW are targeted, providing both CD3-zeta and CD28 intracellular signaling. [Figure 5]The current CARs (A, B, C, D) shown in the literature are shown in comparison with the DuoCARs (E, F, G) according to the present invention. CAR expression vectors can be constructed that induce the expression of a single binding domain (a pair of black, white, or striped circles, each with different specificities) bound to a linker / transmembrane domain (individual white squares). The thick gray line in Figure a represents the plasma cell membrane. In this figure, a pair of black circles may represent anti-CD19 scFv, a pair of white circles may represent anti-CD20 scFv, and a pair of striped circles may represent anti-CD22 scFv, all of which are linked by a linked amino acid sequence, such as a multimer of GGGGS (1, 2, 3, 4, 5, or 6 repeats). Within the cell, lymphocyte signaling domains derived from 4-1BB (CD137), CD28, and CD3-zeta chains can be bound as shown. (A) In a single CAR, one binding domain binds to a transmembrane and two signaling domains, creating a second-generation CAR. (B) In a split CAR, two different binders are expressed along with one signaling domain that must bind to produce effective T cell signaling when recognizing two distinct antigens. (C) In a tandem CAR, two binding domains bind to one signaling domain. In this case, binding to either domain induces sufficient T cell activation. (D) In ​​multiple CARs from a single vector, two fully functional CARs are expressed from a single vector, each capable of binding to only one antigen. (E) On the other hand, a DuoCAR consists of two vectors, expresses at least three binding domains, and has multiple possible combinations of signaling domains. Essential features that distinguish a DuoCAR are the expression of two or more transcripts, the presence of multiple binding domains (at least one of which targets multiple targets), and the presence of at least one of the two expressed cell surface proteins having fully functional signaling properties.(F) In the DuoCAR monospecific soluble binder configuration, the CAR portion encoded by the vector expresses a tag or anti-tag motif that also encodes transmembrane and intracellular signaling motifs (the CAR-based vector and intracellular motif are not identical). The base vector binds to a soluble protein containing both an scFv domain that interacts with the antigen and a tag or anti-tag motif, mediating its binding to the CAR-based protein. When the soluble protein binds to the CAR-based protein, the same structural features [same as in (E)] that mediate the DuoCAR-mediated antitumor activity are reconstituted. (G) In the bispecific soluble binder configuration of DuoCAR, the bispecific "tag"-"anti-tag" interaction is unique, so only one of the soluble binders can bind to only one of the base vectors. In this case, the black rhomboid on the base vector and the mountain-shaped binder on the soluble dual scFv protein may exhibit a "biotin"-"anti-biotin" interaction, and the black crescent shape on the second CAR-based vector may interact with the black oval on the single-specific scFv structure to exhibit a "FITC"-"anti-FITC" interaction. [Figure 6] This study shows the cell surface expression levels of CAR constructs on primary human T cells transduced with different CAR expression vectors in a second-generation (two-co-stimulus domain) and third-generation (three-co-stimulus domain) configuration. T cells were transduced to express the following CARs: no CAR (sham), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and other second-generation CARs (CAR-A-BBz). CAR surface expression levels were detected by flow cytometry and are shown as mean fluorescence intensity (MF) on the y-axis. All constructs expressed identical CAR-binding domains, but the MFI of the two second-generation CARs was significantly brighter. [Figure 7]This shows the cell surface expression of DuoCAR in human T cells. Human T cells were activated in the presence of IL-2 using a CD3-CD28 nanomatrix (TransAct, Miltenyi Biotec), transduced with two vectors (one encoding a tandem CD20-CD19 CAR and the other encoding a single CD22 CAR, thus forming a 2+1 Duo set), and then analyzed for the expression of CD19-, CD20-, or CD22-scFv domains by flow cytometry using recombinant CD19, CD20, or CD22 for staining. In paired columns, the left column shows double staining of CD20 scFv and CD19 scFv, and the right column shows double staining of CD22 scFv and CD19 scFv. The first row shows untransduced T cells (UTD), thus demonstrating the lack of binding. The second panel shows T cells transduced with a LV encoding the CD20_CD19 CAR vector (20-19-28z), which has a CD8 transmembrane domain and intracellular CD28 and CD3-zeta signaling domains. Double staining for CD20 and CD19 binding is observed (left panel), but only CD19 binding is observed in the right panel. The third panel shows T cells transduced with a CD22 CAR vector (22-BBz), which has a CD8 transmembrane domain and intracellular 4-1BB and CD3-zeta signaling domains. No double staining for CD19 or CD20 is observed (left panel), indicating that only one cell population was able to bind to CD22 (right panel). The fourth panel shows T cells transduced with a Duo set containing both the second and third vectors. Only this Duo set expresses all three CAR coding binding domains (42% of cells express CD20_19 (left panel), and 38% express CD22 and CD19 binding domains (right panel)). Since CD22 and CD19 scFv are present in each of the two distinct transmembrane proteins containing the Duo set, the 38% represents a population that truly expresses the Duo set in this example. [Figure 8]The antitumor cell lytic activity of T cells expressing DuoCAR is demonstrated. As shown in Figure 7, human T cells transduced with a single CAR element (20_19-28z or 22-BBz) or DuoCAR (20_19-28z + 22-BBz) were used in a cytotoxic T cell assay at four different effector target ratios (as shown, 20:1, 10:1, 5:1, and 2.5:1). The leukemia cell lines used as CAR-T targets were: Raji (expressing all three target antigens), REH (expressing all three target antigens), K562 (control, no target expression), K562-CD19 (expressing CD19), K562-CD20 (expressing CD20), and K562-CD22 (expressing CD22). Only DuoCAR transduced cells (20-19-28z + 22-BBz, 2+1 Duo set) exhibited high cytolytic activity against both leukemia cell lines (Raji and REH) and all three mono-expressing K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). [Figure 9]This shows DuoCAR cell surface expression in primary human T cells achieved by two different LV preparation methods. Using the same methods and data analysis as in Figure 7, we generated cells transduced with CD19, CD20, and CD22-specific DuoCARs (a 2+1 Duo set consisting of one CAR with a tandem CD20 and CD19 binder and a second CAR with a CD22 binder). The first column of the data shows the expression of CD19 and CD20 binders analyzed by flow cytometry, and the second column shows the expression of CD22 and CD19 binders present as CARs in DuoCAR-expressing cells analyzed by flow cytometry. Four distinct populations—a population without transduction, a population transduced with a single CD22-CAR, a population transduced with dual CD22 and CD20_19 CARs, and a population transduced with a single tandem CD20_CD19 CAR—correspond to the lower left, upper left, upper right, and lower right quadrants, respectively. Both transduction methods using two LVs (simultaneous transduction) and transduction methods using one LV (simultaneous transfection) showed similar DuoCAR staining patterns, and more than 30% of the T cell population was specific to CD19, CD20, and CD22 because it expressed both CAR cell surface proteins. [Figure 10] A schematic diagram of the DuoCAR bicistronic construct is shown. The DuoCAR construct, containing the sequences of two CAR chains separated by a 2A peptide, is expressed from a single bicistronic open reading frame. One CAR consists of a CD22 scFv bound in-frame to a CD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta-activating domain. The other CAR consists of a targeting domain based on a tandem CD20 CD19 scFv, followed by a CD8 hinge / transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta-activating domain. [Figure 11]This shows the cell surface expression of Set 1 bicistronic DuoCAR on primary human T cells transduced with DuoCAR expression vectors and controls, measured by flow cytometry. T cells were transduced to express the following CARs: no CAR (UTD), construct number 2228 (2019 tandem CAR), construct numbers 2200, 2209, 2218, 2225, 2227 (CD22 CAR variant), construct numbers 2515, 2520, 2521 (bicistronic CAR containing one CAR chain targeting CD22 and another tandem CAR chain targeting tumor antigens CD20 and CD19). In the two-part plot shown, CAR 22 expression is shown on the Y-axis, and CAR 19 expression representing the tandem 2019 CAR chain is shown on the X-axis. The percentage of positive cells is shown in each quadrant. The data represents three transduction experiments using T cells from different healthy donors. [Figure 12] This shows the cytokine response of bicistronic DuoCAR set 1 incubated with Raji tumor cells. T cells were transduced to express the following CARs: no CAR (UTD), construct number 2228 (-2019 tandem CAR), construct number 2200 (-CD22 CAR), construct numbers 2515, 2520, and 2521. DuoCAR T cells and controls were incubated overnight with triply positive Raji cells, and the supernatant was collected and analyzed by ELISA for IFNg, TNFα, and IL-2. N=3, ±SD. Each experiment represents three separate experiments with T cells from separate donors. [Figure 13]This shows the cell surface expression of Set 2 bicistronic DuoCARs on primary human T cells transduced with DuoCAR expression vectors and controls, measured by flow cytometry. T cells were transduced to express the following CARs: no CAR (UTD), construct number 1497 (-2019 tandem CAR), construct number 2200 (-CD22 CAR), and construct numbers D0043, D0044, D0046, and D0047 (bicistronic CARs containing one CAR chain targeting CD22 and another tandem CAR chain targeting tumor antigens CD20 and CD19). In the two-part plot shown, CAR 22 expression is shown on the Y-axis, and CAR 19 expression, representing the tandem 2019 CAR chain, is shown on the X-axis. The percentage of positive cells is shown in each quadrant. The data represent three transduction experiments on T cells from three separate healthy donors. [Figure 14] This study demonstrates the antitumor cell lytic activity of bicistronic DuoCAR-expressing T cells from Set 2. Human T cells transduced with a single CAR element (LTG1497, 20_19-28z, or LTG2200, 22-BBz) or DuoCAR (construction numbers D0043, D0044, D0046, D0047, encoding 20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector target ratios (10:1, 5:1, 2.5:1 as shown; zeros between "D" and numerical names in the construct names have been omitted for simplicity). The leukemia cell lines used as CAR-T targets were: Raji (expressing all three target antigens), Reh (expressing all three target antigens), and 392T (not containing any of the three target antigens). DuoCAR lysed triply positive cell lines in an E:T-dependent manner, but did not lyse target-negative 293T cell lines. [Figure 15]This study demonstrates the antitumor cell lysis activity of T cells expressing the bicistronic DuoCAR set 2. Human T cells transduced with a single CAR element (LTG1497, 20_19-28z, or LTG 2200, 22-BBz) or DuoCAR (construction numbers D0043, D0044, D0046, D0047, encoding 20_19-28z + 22-BBz) were used in a cytotoxic T cell assay at four different effector target ratios (10:1, 5:1, 2.5:1 as shown; zeros between "D" and numerical names in the construct names have been omitted for simplicity). The single-positive tumor cell lines used as CAR-T targets were: K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22). Three single-positive tumor cell lines were generated by stable transduction of a single desired antigen (CD19, CD20, or CD22) and the firefly luciferase gene into a parental K562 erythroleukemia strain that did not originally express CD19, CD20, or CD22. DuoCAR lysed the single-positive cell lines in an E:T-dependent manner, and lysis mediated by CAR controls with incompatible antigen-targeting domains (CAR 22, LTG 2200 vs. K19 and K20, tandem CAR 2019, LTG 1479 vs. K22) did not exceed background levels. [Figure 16]This study demonstrates the cytokine response of bicistronic DuoCAR version 2 incubated with Raji tumor cells or in the absence of tumor cells (CAR alone). T cells were transduced to express the following CARs: no CAR (UTD), construct number 2273 (-2019 tandem single-stranded CAR), construct number 2200 (CD22 single-stranded CAR), construct numbers D44, D47 (for each of the D0044 and D0047 CAR constructs, the zero between "D" and the numerical name in the construct name has been omitted for simplicity). DuoCAR and T cells and controls were incubated overnight with tri-positive Raji cells, and the supernatant was collected and analyzed by ELISA for IFNg, TNFα, and IL-2. N=3, ±SD. One experiment represents three separate experiments with T cells from separate donors. [Figure 17] A schematic diagram shows two CAR chains that can be combined for co-expression in the same cell or cell population for the purpose of creating DuoCARs by simultaneous transfection or simultaneous transduction. One CAR chain consists of a CD22 scFv bound in-frame to a CD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta-activating domain. The other CAR chain consists of a targeting domain based on a tandem CD20 CD19 scFv, followed by a CD8 hinge / transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta-activating domain. [Figure 18]This shows flow cytometry measurements of DuoCAR and control cell surface expression on primary human T cells transduced with a DuoCAR expression vector preparation prepared by simultaneous transfection of two transfer plasmids for LV production, or transduced individually with a single vector control (upper panel). T cells were transduced to express the following CARs: construct numbers 2273, 2228 (-2019 tandem CAR), D1, D2, D3, CD22 CAR, and DuoCAR (construct numbers D1+2273, D2+2273, D3+2273). In the scatter plot, CAR 22 expression is shown on the Y-axis, and CAR 19 expression, representing the tandem 2019 CAR chain, is shown on the X-axis. The percentage of positive cells is shown in each quadrant. These are representative data from three experiments using T cells from three donors. [Figure 19A]The study demonstrated antitumor cell lysis activity of DuoCAR cells or single-stranded CAR controls. These DuoCAR T cells were generated by simultaneously transfecting two transfer plasmids to create a lentiviral vector. T cells were transduced using the resulting DuoCAR vector or single-stranded CAR control to express the following CARs: construct number 2273 (-2019 single-stranded tandem CAR); construct numbers D1, D2, D3 (-CD22 single-stranded CAR); and DuoCARs generated by combining two single CAR strands within the same CAR T product (construct numbers D1+2273, D2+2273, D3+2273; the "D" in the name is omitted for simplification). The obtained CAR T cells were analyzed in a cytotoxic T cell assay against native leukemia strains (CD19+CD20+CD22+(Raji, Reh) or CD19, CD20, CD22 triple-negative control strain 293T) at two different effector target ratios (as shown, 10:1 and 5:1) (Figure 19A). The native target strains Raji and Reh were lysed by the single-stranded CAR construct, all DuoCAR groups (construction numbers D1+2273, D2+2273, D3+2273; the "D" in the names has been omitted for simplicity), and the single-stranded CAR control. In contrast, the DuoCAR and single-stranded CAR control did not show cytolysis against CD19, CD20, CD22 triple-negative strain 293T, demonstrating the target specificity of the CAR constructs. Because DuoCAR simultaneously targets three target antigens, and to further address the challenge of target specificity, DuoCAR was tested on recombinant single-positive tumor cells generated from K562 erythroleukemia cells that do not originally express CD19, CD20, or CD22. The single-positive tumor cell lines used as CAR-T targets were: K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22), Figure 19B.DuoCAR lysed single-positive cell lines in an E:T-dependent manner, demonstrating that all targeting domains of DuoCAR are functional and specific to congeneral target molecules (Figure 19B). Furthermore, single-chain CAR controls with incompatible antigen targeting domains (CAR 22, LTG 2200 vs. K19 and K20; tandem CAR 2019, LTG 1479 vs. K22) did not exhibit specific lytic activity (Figure 19B). [Figure 19B]The study demonstrated antitumor cell lysis activity of DuoCAR cells or single-stranded CAR controls. These DuoCAR T cells were generated by simultaneously transfecting two transfer plasmids to create a lentiviral vector. T cells were transduced using the resulting DuoCAR vector or single-stranded CAR control to express the following CARs: construct number 2273 (-2019 single-stranded tandem CAR); construct numbers D1, D2, D3 (-CD22 single-stranded CAR); and DuoCARs generated by combining two single CAR strands within the same CAR T product (construct numbers D1+2273, D2+2273, D3+2273; the "D" in the name is omitted for simplification). The obtained CAR T cells were analyzed in a cytotoxic T cell assay against native leukemia strains (CD19+CD20+CD22+(Raji, Reh) or CD19, CD20, CD22 triple-negative control strain 293T) at two different effector target ratios (as shown, 10:1 and 5:1) (Figure 19A). The native target strains Raji and Reh were lysed by the single-stranded CAR construct, all DuoCAR groups (construction numbers D1+2273, D2+2273, D3+2273; the "D" in the names has been omitted for simplicity), and the single-stranded CAR control. In contrast, the DuoCAR and single-stranded CAR control did not show cytolysis against CD19, CD20, CD22 triple-negative strain 293T, demonstrating the target specificity of the CAR constructs. Because DuoCAR simultaneously targets three target antigens, and to further address the challenge of target specificity, DuoCAR was tested on recombinant single-positive tumor cells generated from K562 erythroleukemia cells that do not originally express CD19, CD20, or CD22. The single-positive tumor cell lines used as CAR-T targets were: K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22), Figure 19B.DuoCAR lysed single-positive cell lines in an E:T-dependent manner, demonstrating that all targeting domains of DuoCAR are functional and specific to congeneral target molecules (Figure 19B). Furthermore, single-chain CAR controls with incompatible antigen targeting domains (CAR 22, LTG 2200 vs. K19 and K20; tandem CAR 2019, LTG 1479 vs. K22) did not exhibit specific lytic activity (Figure 19B). [Figure 20] DuoCAR cells or single-stranded CAR controls exhibit cytokine release activity in response to Raji13G11, CD19+CD20+CD22+ clones. These DuoCAR T cells were generated by simultaneously transfecting two transfer plasmids to create lentiviral vectors. T cells were transduced using the obtained DuoCAR vectors, and the following CARs were expressed: construct number 2273 (-2019 tandem CAR); construct numbers D1, D2, D3 (-CD22 CAR); and three DuoCARs (D1+2273, D2+2273, D3+2273, Figure 20; the "D" in the group names is omitted for simplicity). The resulting CAR T cells were co-cultured with the triple-positive Raji tumor line at an E:T ratio of 10 overnight, and the culture supernatant was analyzed for IFNg, TNFα, and IL-2. All Duo CAR constructs showed high levels of three cytokines in response to Raji cells. DuoCAR alone, consisting of CAR T cells incubated in the absence of the Raji target, did not show clear cytokine production in response to Raji 13G11 cells, indicating that the cytokine response is target-specific (Figure 20). [Modes for carrying out the invention]

[0082] Detailed explanation definition Where used herein, the singular forms “a,” “an,” and “the” refer to both the singular and plural forms unless there is a clear contextual inconsistency. For example, the term “an antigen” can be considered equivalent to the phrase “at least one antigen,” meaning one or more antigens. Where used herein, the term “comprises” means “includes.” Therefore, “comprising an antigen” means “including an antigen,” without excluding other elements. The words “and / or” also mean “and and "or" means "or". Furthermore, unless otherwise specified, any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values ​​given to nucleic acids or polypeptides, are approximate and provided for illustrative purposes only. Many methods and materials similar to or equivalent to those described herein can be used, but particularly preferred methods and materials are described below. In the event of any inconsistency, this specification (including the definitions of terms) shall prevail. In addition, materials, methods, and examples are for illustrative purposes only and not intended to limit. A definition of terms is provided below to facilitate reference to the diverse embodiments.

[0083] The term "approximately" means, when referring to measurable values ​​such as quantity and duration, to include variables within ±20%, ±10%, or more preferably ±5%, or ±1%, or even more preferably ±0.1%, of the explicitly stated value, because such variables are appropriate for carrying out the disclosed method.

[0084] Unless otherwise specified, scientific terms used herein are used in their conventional sense. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0085] The present invention relates to a patient-specific and tumor-specific method for transferring two or more vector-transduced T cells into adoptive cells to express one or more DuoCARs.

[0086] More specifically, the present invention relates to a lentiviral vector expressing a chimeric antigen receptor (DuoCAR), and also provides, for example, a host cell (e.g., lymphocyte, T cell) transduced with the lentiviral vector expressing the CAR, a nucleic acid molecule encoding the lentiviral vector and the chimeric antigen receptor, and methods of using them, for example, to treat cancer in a subject.

[0087] Surprisingly and unexpectedly, the inventors have found that an immunotherapy composition containing a patient-specific autologous antitumor lymphocyte cell population exhibits significantly increased efficacy as an antitumor immunotherapy when two or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs) are transduced into the autologous lymphocyte cell population. The use of at least two lentiviral vectors expressing one or more CARs promotes the in vivo proliferation and persistence of patient-specific antitumor T cells, and As a result, it appears to bring about patient-specific outcomes such as tumor stabilization, reduction, removal, or remission of cancer, or prevention or remission of cancer recurrence, or any combination thereof.

[0088] Such active patient-specific antitumor T cell populations described herein can be returned to the patient by direct injection to promote the in vivo proliferation and persistence of patient-specific antitumor T cells, resulting in patient-specific stabilization, reduction, or removal of tumors, remission of cancer, or prevention or remission of cancer recurrence, or a combination thereof. This also includes effective proliferation and rapid contraction of the therapeutic cell population.

[0089] Therefore, in its broadest sense, the novelty of this adoptive immunotherapy lies in the use of a combination of CAR expression vectors. A key difference is that, unlike the conventional use of a single vector expressing one or more chimeric antigen receptors, the Duo CAR approach yields both multi-antigen specificity and optimal signaling for antitumor T cell activity in vivo. Creating a system in which three or more antigens are efficiently targeted is significantly superior to single- or tandem approaches, which can lead to tumor metastasis and / or recurrence due to the potential for escape mutations by tumor cells. In the use of two or more vectors encoding one or more chimeric antigen receptors (DuoCARs), it is also required that at least one specific combination of binding domains is not identical in each vector, and furthermore, at least one combination of signaling motifs is not identical among each of the vectors, thereby generating a patient-specific autologous antitumor lymphocyte population into which one or more genetically modified lymphocyte populations transduced with such duo lentiviral vector-derived CARs can promote in vivo proliferation and persistence of patient-specific antitumor lymphocytes, and as a result, bring about patient-specific stabilization, reduction, removal, or remission of tumors or cancers, and / or prevention or remission of tumor or cancer recurrence, or any combination thereof.

[0090] In one embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each encoding a functional CAR, wherein at least one binding domain in one of the vectors is not identical, resulting in the expression of two or more non-identical binding domains as a result of the combination of the vectors, each of which is covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0091] In another embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains as a result of the combination of the vectors, each of which binding domains encoded by the vectors is covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs, except that the immunotherapeutic composition excludes single CARs, split CARs, tandem CARs, or multiple CARs as shown in Figures 5(A), (B), (C), or (D), respectively.

[0092] The immunotherapeutic efficacy and prevention or remission of tumor or cancer recurrence achieved using DuoCAR lentiviral vector-modified T cells according to the present invention significantly surpasses, and synergistically surpasses, the results of conventional single-CAR designs. This unique combination of biological therapeutic benefits, coupled with other factors, leads to increased in vivo proliferation and persistence of patient-specific antitumor lymphocytes compared to conventional CAR-based T cell immunotherapy, resulting in tumor or cancer stabilization, reduction, removal, or remission.

[0093] CAR expression vectors can be constructed to induce the expression of a single binding domain (black, white, or striped circles, each with different specificities, Figure 5) bound to the linker domain and transmembrane domain (individual white squares). Figure 5 below shows a comparison of a conventional CAR and the DuoCAR according to the present invention. In Figure 5, the thick gray line represents the plasma cell membrane. Intracellularly, lymphocyte signaling domains derived from 4-1BB (CD137), CD28, and the CD3-zeta chain can be bound as shown. All examples and uses of the CD3 signaling domain in this application involve modifying the CD3 zeta chain by selectively mutagenerating (or otherwise altering) one, two, or three immunoreceptor-activating tyrosine motifs (ITAMs) of tyrosine residues within them. In a single CAR (Figure 5A), one binding domain is bound to the transmembrane domain and two signaling domains. In a split CAR (Figure 5B), two different binders are represented along with one signaling domain to which they must bind in order to produce effective signaling. In a tandem CAR (Figure 5C), two binding domains are bound to one signaling domain. In multiple CARs from a single vector (Figure 5D), two fully functional CARs are expressed from a single vector. A Duo-CAR according to the present invention (for example, Figure 5E) encodes at least two vectors, each encoding a functional CAR, resulting in the expression of two or more non-identical binding domains as a result of the vectors being combined, each of which is covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs. Essential features that distinguish a DuoCAR according to the present invention are the use of two or more vectors, the presence of multiple binding domains, and the fact that at least one of the two expressed cell surface proteins has fully functional signaling properties (related to T cell proliferation in vivo).

[0094] In another embodiment, DuoCAR is used to enhance the immune response against tumors mediated by a therapeutic T cell population. The immune response is enhanced in at least three ways.

[0095] Firstly, by providing T cells with further signals for proliferation and survival in the body, the DuoCAR according to the present invention can sustain a therapeutic T cell population by stimulating the T cell population upon encountering an autoantigen (e.g., CD19) (which the patient can tolerate without) (which serves to provide a stimulating signal to the therapeutic cell population that is not present in the tumor tissue itself). Third-generation DuoCARs (expressing three co-stimulatory domains intracellularly and bound to one extracellular Ig-like binder) are well known and have been shown not to be expressed on therapeutic T cells, similar to DuoCARs expressing two intracellular co-stimulatory domains. For example, in Figure 6 below, the expression level of the CAR construct on primary human T cells differs between the second-generation (two co-stimulatory domains) construct and the third-generation (three co-stimulatory domains) construct. T cells were transduced to express the following CARs: no CAR (pseudo), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and other second-generation CARs (CAR-A-BBz). Surface expression levels of the CARs were detected by flow cytometry and are shown as mean fluorescence intensity (MF) on the y-axis. Although all constructs expressed identical CAR-binding domains, the MFI of the two second-generation CARs was significantly brighter.

[0096] By providing a third T cell activation sequence to another vector CAR construct, the inventors can achieve the advantage of expressing three co-stimulatory domains without the disadvantage of reduced CAR expression on the T cell surface.

[0097] In a second embodiment, the DuoCAR according to the present invention may target non-tumor cell types that mediate the immunosuppressive effect. For example, if CD19-expressing B cells are present in a tumor lesion and inhibit anti-tumor immunity by producing IL-4 or other mediators, a second benefit of using a tumor-specific T cell population expressing DuoCAR is that this immunosuppressive cell population is also eliminated.

[0098] For example, if immunosuppressive B cells are present in a solid tumor lesion, they can be eliminated using a B cell-specific DuoCAR (such as a CD19-specific DuoCAR). If immunosuppressive fibroblast-like cells are present, they can be eliminated using a stromal-specific DuoCAR (for example, by targeting fibroblast-activating protein alpha (FAP)). If an effective immune response is impaired due to vascular malformations, treatment outcomes can also be improved with a DuoCAR specific to such types of blood vessels or lymphatic vessels (such as a VEGFR antibody).

[0099] In a third embodiment, the DuoCAR according to the present invention targets immunosuppressive populations distal to the tumor (i.e., located in a different compartment of the body). For example, a DuoCAR is used that targets myeloid-derived suppressor cells (MDSCs) that may be present in the tumor lesion itself or in local lymph nodes or bone marrow. Tumor-draining lymph nodes have been shown to be sites of both immune activation and immunosuppression. This depends on the overall degree of inflammation in the lymph node and distal dendritic cell differentiation that occurred before migration to the lymph node. If antigen-presenting cells such as myeloid-derived suppressor cells (MDSCs) or misdifferentiated dendritic cells are present in tumor-draining lymph nodes, treatment outcomes can be improved using DuoCARs that target these cell types (even distal to the tumor itself). In addition to cancer-specific DuoCAR immunotherapy applications, a second application of DuoCAR is the prevention or treatment of autoimmune and / or inflammatory diseases. The difference from oncology applications is that T regulatory cells (Treg), or inducible T regulatory cells (iTreg), or other cells cultured under conditions that promote Th-2-like immune responses can serve as the cellular matrix. In oncology applications, Th-1-like cells are the cellular matrix. In a wide range of therapeutic applications, such as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT), allergic respiratory, intestinal, or other mucosal inflammation, or skin allergies, the presence of CAR-modified lymphocytes that produce immunosuppressive cytokines such as transforming growth factor beta (TFG-beta) is thought to generate a broad range of immunotolerogenic signals that lead to remission of autoimmune or inflammatory diseases. This approach includes neurological inflammatory conditions of the peripheral or central nervous system (CNS), such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, Parkinson's disease, and CTE (chronic traumatic encephalopathy caused by repeated concussions or microconcussions). Furthermore, this approach also applies to progressive scarring diseases such as COPD (chronic obstructive pulmonary disease).

[0100] In the treatment of inflammatory diseases, lymphocytes specific to tissue antigens, or stress markers on the surface of inflamed cells, or misfolded proteins (such as tau protein or beta-amyloid), can be produced by generating DuoCAR expression vectors specific to these targets. Single-antibody-based Alzheimer's disease therapies are already in clinical use (i.e., solanezumab by Eli Lilly and aducanumab by Biogen). In Alzheimer's disease, antibodies against monomeric or aggregated beta-amyloid can be used in the CAR manner instead of binders that bind to cell surface proteins. Binders that bind to MHC molecules-bound tau protein or tau peptides can also be used as CAR binding motifs. Furthermore, receptors that mediate lymphocyte homing to specific peripheral tissues can be included in the CAR manner to provide local specificity to CAR-expressing Treg populations. It is known that lymphocytes infiltrate specific tissues. Adhesion receptor domains and cytokine sequences or cytokine or chemokine receptors or binders can be used as part of the CAR domain. Adhesion molecules such as CD44 and integrin alpha-4 are known to direct lymphocytes to the CNS, and therefore, it is conceivable that CAR-expressing lymphocytes can be directed to disease sites using domains derived from adhesion molecules known to mediate the CNS migratory behavior of lymphocyte populations. The same is thought to be true for the intestines (i.e., expression of binders that bind to MAdCAm-1, CCR9, or anti-CCL25 antibodies), the lungs (i.e., P-selectin or mesothelin), the skin (i.e., binders that bind to E-selectin), or other mucosal surfaces.

[0101] To employ this approach, patients with inflammatory conditions or diseases treatable by reducing inflammatory states (such as Alzheimer's disease) can be admitted to a clinic for peripheral blood collection. Tregs can be directly selected using immunomagnetic beads (regulatory T cell isolation kit, Miltenyi Biotec) or induced by culture under appropriate cytokine conditions. These Tregs or iTregs can then be transduced using a DuoCAR vector and, if necessary, proliferated in vitro (Treg proliferation kit, Miltenyi Biotec). The DuoCAR-binding domain may be derived from an antibody, a receptor mediating tissue-specific homing, or a disease-related binder such as anti-beta-amyloid. These manipulated immunoeffector cells can then be directed to appropriate sites to produce cytokines corresponding to their Th2 or Treg differentiation pattern. It is also known that CAR-T cells can be manipulated to secrete specific gene payloads when the CAR receptor is activated. In addition to the DuoCAR payload expressed from the vector, manipulation of the T cell population can lead to the expression or secretion of further therapeutic proteins or peptides, such as a) A-beta DP (amyloid-beta degrading protease), b) matrix proteases (e.g., MMP-9 and MMP9 inhibitors in COPD), c) peptides or soluble antibody-like binders that inhibit plaque formation, and d) cytokines (e.g., TGF-beta, IL-4, IL-10).

[0102] Furthermore, it is possible to express miRNAs that regulate T cell function within cells. Examples of miRNAs include miR-92a, miR-21, miR-155, miR-146a, miR-3162, miR-1202, miR-1246, and miR-4281, miR-142, and miR-17-92. It is also possible to create shRNAs targeting miRNAs. Examples include shRNAs that target miR-28, miR-150, and miR-107, which generally bind to PD1 and increase its expression.

[0103] Beyond its applications in oncology and inflammatory and autoimmune diseases, a third application of Duo CAR technology is the generation of therapeutic lymphocyte populations specific to viral, bacterial, or fungal antigens. Thus, in the oncological applications described for B-cell malignancies, Duo CAR products can be intervened in immunoprotective or immunotherapeutic activity against infectious pathogens or diseased tissues by targeting infections and enabling recognition of microbial antigens. Unlike T-cell receptor (TCR)-based approaches, where the cell receptor itself is involved in the recognition of pathogen-encoded peptides, the Duo CAR approach utilizes binding proteins expressed in a CAR vector manner, allowing the transduced T cell population to recognize antibodies (i.e., without antigen processing). When the therapeutic T cell population is activated, it can result in an effective immune response against infectious pathogens, as immune activation loci can eliminate infected cells and release soluble mediators such as interferon-gamma if microbial antigens are not bound to the cells.

[0104] For example, HIV is known to be highly mutable, however, it is possible to classify it into specific clades or families and produce antibodies against clade-specific viral envelope proteins (env, gp120). Using the DuoCAR approach, broad anti-HIV immunoactivity can be obtained by including three or more clade-specific antibody-like binders in a CAR construct. In addition to viral proteins, bacterial proteins can also be targeted. A current medical challenge is the treatment of antibiotic-resistant bacterial strains that frequently occur in clinical settings. These include VRE (vancomycin-resistant enterococci), MRSA (methicillin-resistant Staphylococcus aureus), and KPC (Klebsiella pneumoniae carbapenemase-producing Gram-negative bacteria, CRKP). Klebsiella cell surface antigens include O antigens (9 variants) and K antigens (approximately 80 variants). The O antigen spectrum can be easily handled with a small DuoCAR library, and the same is true for multiple K antigens. For use, CAR constructs featuring antibodies that bind to different K or O serotypes can be created, and these CAR vectors can be used to transduce, isolate, and activate Th1-like effector cell populations, as in oncological applications. In fungal diseases, L. Cooper et al.'s research (Kumasesan, PR, A study (2014, PNAS USA, 111:10660) showed that dectin-1, a fungal-binding protein normally expressed on human cells, can be reconstituted as a CAR, and that this can be used to control fungal growth in vitro. Aspergillosis, a human disease, occurs in severely immunosuppressed patients and is caused by the fungus A. fumigatus. Several groups have created monoclonal antibodies specific to antigenic components on the surface of Aspergillus cells, opening the door to adoptive immunotherapy using DuoCARs that target three or more Aspergillus antigens on the fungal surface. This has made it possible to create immunoglobulin-like binders that bind to microbial antigens in all of these infectious disease applications, making it possible to target multiple antigens with a population of CAR-expressing effector lymphocytes.

[0105] Next, DuoCAR, which may be used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein, will be described in detail. This description will include a description of its extracellular domain, transmembrane domain, and intracellular domain, and further descriptions of DuoCAR, antibodies, their antigen-binding fragments, conjugates, nucleotides, expression, vectors, and host cells, as well as methods of treatment, compositions, and kits using the disclosed DuoCAR. Up to this point, compositions and methods according to the present invention have been illustrated with reference to the preparation and use of DuoCAR, but in particular, it is intended herein that such compositions and methods include the preparation and use of TrioCAR and QuatroCAR.

[0106] A. Chimeric antigen receptor (present in DuoCAR) The DuoCAR disclosed herein comprises at least two vectors, each encoding a functional CAR, resulting in the expression of two or more non-identical binding domains as a result of the vectors being combined, each of which is covalently bound to a transmembrane domain and one or more non-identical intracellular signaling motifs, and at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0107] CARs are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain (e.g., a single-strand variable fragment (scFv)) of an antibody bound to a T cell signaling domain via a transmembrane domain. DuoCAR features include the ability to redirect T cell specificity and responsiveness to selected targets in an MHC-independent manner, leveraging the antigen-binding properties of monoclonal antibodies. Because they can recognize antigens independently of MHC, T cells expressing DuoCARs can perform antigen processing independently of antigen processing. It has the ability to recognize antigens and, as a result, evade the main mechanism of tumor escape. Furthermore, when expressed on T cells, DuoCAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0108] As disclosed herein, the intracellular T cell signaling domain of DuoCAR may include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of the CAR that includes the intracellular domain of a T cell receptor, such as (but not limited to) the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand necessary for lymphocytes to respond efficiently to an antigen. In some examples, the activating domain may be attenuated by mutations at specific sites of phosphorylation (i.e., the ITAM motif in the CD3 zeta chain), thereby allowing careful modulation of the degree of signaling mediated by this domain.

[0109] 1. Extracellular domain In one embodiment, the CAR used in the patient-specific autologous antitumor lymphocyte cell population disclosed herein includes a target-specific binding element, also referred to as an antigen-binding domain or site. The selection of the domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease condition. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in a CAR include those associated with viral infections, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0110] In one embodiment, a CAR can be designed to target a desired tumor antigen by designing a desired antigen-binding domain that specifically binds to the antigen on tumor cells. Tumor antigens are proteins produced by tumor cells that trigger an immune response, particularly a T-cell-mediated immune response. The selection of the antigen-binding domain may depend on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigens (CEAs), beta-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I receptor, IGF-II receptor, IGF-I receptor, and mesothelin. The tumor antigens disclosed herein are included for illustrative purposes only. Their listing is not intended to limit them, and other examples will be readily apparent to those skilled in the art.

[0111] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express multiple proteins that can act as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules include transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens includes carcinoembryonic antigens such as carcinoembryonic antigens (CEAs). In B-cell lymphomas, tumor-specific idiotype immunoglobulins... These are truly tumor-specific immunoglobulin antigens, which are unique to each individual tumor. B-cell differentiation antigens such as CD19, CD20, CD22, and CD37 are also candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets in passive immunotherapy using monoclonal antibodies, but have not been sufficiently successful.

[0112] The type of tumor antigen may be either a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are specific to tumor cells and do not occur on other cells in the body. TAAs are not specific to tumor cells and, instead, are expressed on normal cells under conditions that do not induce immune tolerance to this antigen. The expression of this antigen in tumors may occur under conditions that allow the immune system to respond to this antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is not yet mature and cannot respond to the antigen, or TAAs may be antigens that are normally present at very low levels on normal cells but are expressed at significantly higher levels on tumor cells.

[0113] Examples of TSAs or TAAs include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; intrinsic tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as the Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA Includes 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0114] In one preferred embodiment, the antigen-binding domain portion of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, and MAGE A3 TCR. In yet another embodiment, a DuoCAR comprising a tag or anti-tag binding domain is provided herein.

[0115] Depending on the desired target antigen, the CAR may be designed to include an appropriate antigen-binding domain specific to that desired antigen target. For example, if CD19 is the desired target antigen, an antibody against CD19 or a CD19-specific scFv subfragment may be used as the antigen-binding domain to be incorporated into the CAR.

[0116] In one exemplary embodiment, the antigen-binding domain of the CAR targets CD19. Preferably, the antigen-binding domain in the CAR is anti-CD19 scFV, In this embodiment, the nucleic acid sequence of anti-CD19 scFV includes the sequence of SEQ ID NO: 27. In one embodiment, anti-CD19 scFV includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the anti-CD19 scFV portion of CAR includes the amino acid sequence of SEQ ID NO: 28. In a second exemplary embodiment, the antigen-binding domain of CAR targets CD20. Preferably, the antigen-binding domain in CAR is anti-CD20 scFv, where the nucleic acid sequence of anti-CD20 scFv includes the sequence of SEQ ID NO: 1. In another embodiment, the anti-CD20 scFV portion of CAR includes the amino acid sequence of SEQ ID NO: 2. In a third exemplary embodiment, the antigen-binding domain of CAR targets CD22. Preferably, the antigen-binding domain in CAR is anti-CD22 scFv, where the nucleic acid sequence of anti-CD22 scFv includes the sequence of SEQ ID NO: 7. In another embodiment, the anti-CD22 scFV portion of CAR includes the amino acid sequence of SEQ ID NO: 8.

[0117] In one aspect of the present invention, a CAR capable of binding to anything other than TSA or TAA is provided, comprising, for example, an antigen (not limited to) derived from the Retroviridae family (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), the Picornaviridae family (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae, herpesviridae family (e.g., herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesviruses), the Poxviridae family (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0118] In another aspect of the present invention, a CAR capable of binding to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella is provided. In particular, for example, Helicobacter pylori, Legionella pneumophila, and Mycobacterium species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare). M. kansaii or M. gordonea), yellow grape CARs are provided that can bind to antigens derived from infectious bacteria such as cocci, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocystis, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactia), Streptococcus pneumoniae, or Neisseria tetanus, or combinations thereof.

[0119] 2. Transmembrane domain In a DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population disclosed herein, the CAR comprises one or more transmembrane domains fused to the extracellular domain of the CAR.

[0120] In one embodiment, an isolated nucleic acid molecule is provided, in which the encoded linker domain is derived from the extracellular domain of CD8 and bound to the transmembrane domain.

[0121] In one embodiment, an isolated nucleic acid molecule is provided, in which the encoded linker domain originates from and is bound to the extracellular domain of the transmembrane domain.

[0122] In some cases, to minimize interaction with other components of the receptor complex, the transmembrane domain is transmembrane of surface membrane proteins of the same or different type. They may be selected or substituted with amino acids to avoid binding to the domain.

[0123] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention may originate from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, Fc epsilon R, or any combination thereof (i.e., including at least these transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it may mainly consist of hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker, preferably 2 to 10 amino acids in length, may form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A doublet or triple alanine motif of glycine and serine is a particularly suitable linker.

[0124] In one embodiment, the transmembrane domain in the CAR according to the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain includes the nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the CD8 transmembrane domain includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12. In another embodiment, the CD8 transmembrane domain includes the amino acid sequence of SEQ ID NO: 12.

[0125] In some examples, the transmembrane domain of the CAR includes a CD8 alpha-hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO: 13. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. In another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 14.

[0126] While not intended to limit to any specific mechanism of action, reasons for improved therapeutic function in relation to the exemplary DuoCARs of the present invention used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein may include, but are not limited to, the following: a) improved lateral movement within the cell membrane leading to increased signaling efficiency; b) superior positioning within cell membrane microdomains (e.g., lipid rafts) leading to improved interaction with transmembrane signaling cascades associated with T cell activation; c) superior positioning within the cell membrane due to a preference for movement away from reducing or downregulating interactions, such as being relatively far from or having relatively little interaction with phosphatases such as CD45; and d) superior binding to T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0127] In one embodiment of the patient-specific autologous antitumor lymphocyte cell population disclosed herein, the non-limiting and exemplary transmembrane domains used in the DuoCAR disclosed herein include TNFRSF16. As disclosed in the concurrently pending Provisional Patent Application No. 62 / 239,509 (titled "Chimeric Antigen Receptor and Method of Use," filed October 9, 2015, Lentigen Technology, case number LEN_015PRO) by the same applicant, the TNFRSF19 transmembrane domain may be used to induce the TNFRSF transmembrane domain and / or linker domain or spacer domain (in particular, the tumor necrosis factor receptor superfamily listed in Table I thereof). (Including other TNFRSF components listed below).

[0128] 3. Spacer Domain In DuoCARs used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein, a spacer domain may be located between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. A spacer domain means any oligopeptide or polypeptide that functions to bind the TNFRSF transmembrane domain to the extracellular domain and / or the TNFRSF transmembrane domain to the intracellular domain. The spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0129] In some embodiments, the linker may include a spacer element, which, when present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art, as seen in U.S. Patents 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521,2 Including Patent Nos. 84, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (all of which are incorporated herein by reference).

[0130] The spacer domain preferably has a sequence that promotes the binding of the CAR to the antigen and increases signaling into the cell. Examples of amino acids expected to promote binding include cysteine, charged amino acids, and serine and threonine at sites where glycosylation is possible, and these amino acids can be used as amino acids constituting the spacer domain.

[0131] As this spacer domain, all or part of amino acids 137-206 (SEQ ID NO: 15), including the hinge region of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP__000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP__006130.1) can be used. Alternatively, a portion of the constant region of the antibody's H chain or L chain (CH1 region or CL region, for example, a peptide with the amino acid sequence of SEQ ID NO: 16) can also be used as this spacer domain. Furthermore, this spacer domain may be an artificially synthesized sequence.

[0132] Furthermore, a signal peptide sequence may be bound to the N-terminus of the CAR. This signal peptide sequence is present at the N-terminus of many secretory and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above have a signal peptide sequence as an intracellular domain, this signal peptide can be used as a signal peptide for the CAR. In one embodiment, the signal peptide includes the nucleotide sequence of the leader (signal peptide) sequence of SEQ ID NO: 5. In another embodiment, the signal peptide includes the amino acid sequence of SEQ ID NO: 6.

[0133] 4. Intracellular domains The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to a specific function of the cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the protein portion that transmits effector function signals and prompts the cell to perform its specific function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. If a cleaved portion of the intracellular signaling domain is used, it may be used in place of the complete chain as long as this cleaved portion can transmit effector function signals. Therefore, the meaning of the term "intracellular signaling domain" includes any cleaved portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0134] Preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that cooperate to initiate signaling after antigen-receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0135] It is known that signals transmitted solely through the TCR are insufficient to fully activate T cells, and that a second or co-stimulatory signal is required. Therefore, T cell activation can be said to involve two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent activation via the TCR (first cytoplasmic signaling sequence), and another that acts independently of the antigen to provide a second or co-stimulatory signal (second cytoplasmic signaling sequence).

[0136] The first cytoplasmic signaling sequence regulates the first activation of the TCR complex in either a stimulating or inhibitory manner. The first cytoplasmic signaling sequence that functions in a stimulating manner may contain a signaling motif known as an immunoreceptor-activated tyrosine motif or ITAM.

[0137] Examples of ITAMs containing a first cytoplasmic signaling sequence particularly useful in CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAM include amino acid numbers 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acid numbers 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acid numbers 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acid numbers 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acid numbers 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acid numbers 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), and CD5 (NCBI Amino acid numbers 402-495 of 0022 (NCBI RefSeq:NP__055022.2), amino acid numbers 707-847 of 0022 (NCBI RefSeq:NP__001762.2), amino acid numbers 166-226 of CD79a (NCBI RefSeq:NP__001774.1), amino acid numbers 182-229 of CD79b (NCBI RefSeq:NP__000617.1), and CD66d (NCBI This includes, but is not limited to, peptides having amino acid sequences 177-252 of RefSeq:NP__001806.2), as well as variants having the same function as these peptides. The amino acid numbers based on NCBI RefSeq ID or GenBank amino acid sequence information described herein are those of the precursor (signature) of each protein. These are numbered based on the full length of the sequence (including the sumpeptide sequence). In one embodiment, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling sequence derived from the CD3 zeta. In another embodiment, one, two, or three ITAM motifs in the CD3 zeta are attenuated by mutations in tyrosine residues or substitutions with other amino acids.

[0138] In one preferred embodiment, the intracellular domain of the CAR may be designed to include a CD3-zeta signaling domain by itself, or may be combined with any other desirable cytoplasmic domain useful in association with the CAR. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand necessary for lymphocytes to respond efficiently to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific examples of such co-stimulatory molecules include amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP__001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP__000607.1), amino acid numbers 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acid numbers 196-210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181-220 of CD28 (NCBI RefSeq: NP__006130.1), and CD137 (4-1BB, NCBI This disclosure includes, but is not limited to, peptides having the sequences of amino acid numbers 214-255 of RefSeq:NP__001552.2), amino acid numbers 241-277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and amino acid numbers 166-199 of ICOS (NCBI RefSeq:NP__036224.1), as well as variants having the same function as these peptides. Thus, while this disclosure has primarily illustrated 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of this disclosure.

[0139] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of CAR may be linked to each other in a random or specific order. Optionally, short oligo or polypeptide linkers, preferably 2 to 10 amino acids in length, may form these links. A doublet of glycine and serine provides a particularly suitable linker.

[0140] In one embodiment, the intracellular domain is designed to include the signaling domain CD3-zeta and the signaling domain CD28. In another embodiment, the intracellular domain is designed to include the signaling domain CD3-zeta and the signaling domain 4-1BB. In yet another embodiment, the intracellular domain is designed to include the signaling domain CD3-zeta, as well as the signaling domains CD28 and 4-1BB.

[0141] In one embodiment, the intracellular domain in CAR is designed to include a signaling domain 4-1BB and a signaling domain CD3-zeta, where the signaling domain 4-1BB includes the nucleic acid sequence of SEQ ID NO: 17 and the signaling domain CD3-zeta includes the nucleic acid sequence of SEQ ID NO: 19.

[0142] In one embodiment, the intracellular domain in CAR is designed to include the signaling domain 4-1BB and the signaling domain CD3-zeta, where the signal The signaling domain 4-1BB contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and the signaling domain CD3-zeta contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0143] In one embodiment, the intracellular domain in CAR is designed to include a signaling domain 4-1BB and a signaling domain CD3-zeta, where the signaling domain 4-1BB includes the amino acid sequence of SEQ ID NO: 18 and the signaling domain CD3-zeta includes the amino acid sequence of SEQ ID NO: 20.

[0144] 5. Further explanation about DuoCAR The functional portions of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein are also expressly included within the scope of the invention. When used in reference to CAR, the term “functional portion” means one or more arbitrary portions or fragments of DuoCAR disclosed herein, which retain the biological activity of the CAR (parent CAR). The functional portion includes, for example, a CAR portion that retains the ability to recognize target cells or to detect, treat, or prevent disease to a degree similar to, comparable to, or higher than that of the parent CAR. With respect to the parent CAR, the functional portion may contain, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0145] The functional moiety may contain additional amino acids at its amino-terminus, carboxy-terminus, or both, that are not present in the amino acid sequence of the parent CAR. Preferably, these additional amino acids do not interfere with the biological function of the functional moiety, such as target cell recognition, cancer detection, cancer treatment, or prevention. More preferably, these additional amino acids enhance such biological activity compared to that of the parent CAR.

[0146] Functional variants of DuoCAR disclosed herein are included within the scope of this disclosure. The term “functional variant,” as used herein, means a CAR, polypeptide, or protein having a substantial or significant degree of sequence identity or similarity to a parent CAR, the functional variant retaining the biological activity of the CAR from which the variant is derived. Functional variants include, for example, variants of a CAR (parent CAR) described herein that retain the ability to recognize target cells to a degree similar to, equal to, or greater than that of the parent CAR. With respect to a parent CAR, a functional variant may have, for example, amino acid sequence identity with the parent CAR of at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0147] A functional variant may, for example, include the amino acid sequence of the parent CAR with at least one conserved amino acid substitution. Alternatively, or additionally, a functional variant may include the amino acid sequence of the parent CAR with at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant so that, as a result, the biological activity of the functional variant is superior to that of the parent CAR.

[0148] The amino acid substitutions in DuoCAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property. For example, a conservative amino acid substitution is replacing an acidic / loading electrode amino acid (e.g., Asp or Glu) with another acidic / loading electrode amino acid Substitution may include substitution with an acid, substitution of a nonpolar side-chain-containing amino acid (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another nonpolar side-chain-containing amino acid, substitution of a basic / positively charged amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged amino acid, substitution of a polar side-chain-containing uncharged amino acid (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) with another polar side-chain-containing uncharged amino acid, substitution of a beta-branched side-chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side-chain-containing amino acid, substitution of an aromatic side-chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side-chain-containing amino acid, and so on.

[0149] CAR may essentially consist of one or more designated amino acid sequences described herein, and as a result, the biological activity of the functional variant is not substantially altered by other components (e.g., other amino acids).

[0150] DuoCAR (including functional moieties and functional variants) may be of any length, i.e., may contain any number of amino acids, as long as the DuoCAR (or its functional moiety or functional variant) retains biological activity, such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent a disease in mammals. For example, a CAR may be about 50 to about 5000 amino acids long, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more.

[0151] DuoCAR (including the functional portion and functional variant according to the present invention) may contain synthetic amino acids instead of one or more natural amino acids. Such synthetic amino acids are well known in the art, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentane carboxylic acid It contains (acid), α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0152] DuoCAR (including the functional moiety and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide crosslinking), or converted to an acid addition salt, and / or optionally dimerized, polymerized, or conjugated.

[0153] DuoCAR (including its functional portion and functional variants) can be obtained by methods well known in the art. DuoCAR may be prepared by any suitable polypeptide or protein synthesis method. Suitable methods for the new synthesis of polypeptides and proteins are described in Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis. s, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. 5, 4 This is described in prior art literature such as No. 49,752. Methods for generating chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for the treatment of cancer) are well known in the art and are further described herein (e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., See 6:47, 2013; Tumaini et al., Cytotherapy, 15, 1406-1417, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Public Notice No. 2012 / 0213783 (each of these in its entirety is incorporated herein by reference). For example, the disclosed CARs can also be constructed by including a nucleic acid molecule encoding the disclosed chimeric antigen-binding receptor in an expression vector (such as a lentiviral vector) used for transduction into host cells such as T cells. In some embodiments, the method of using a chimeric antigen receptor includes isolating T cells from a subject, transducing these T cells with an expression vector encoding the chimeric antigen receptor (such as a lentiviral vector), and administering the CAR-expressing T cells to the subject for a treatment such as treating a tumor in the subject.

[0154] B. Antibodies and antigen-binding fragments One embodiment further provides a CAR, a CAR-expressing T cell, an antibody, or its antigen-binding domain or portion (all of which specifically bind to one or more of the antigens disclosed herein) used in a patient-specific autologous antitumor lymphocyte cell population disclosed herein. Where used herein, “CAR-expressing T cell” or “CAR T cell” means a T cell that expresses a CAR and has antigen specificity, for example, determined by the antibody-derived targeting domain of the CAR.

[0155] As used herein, “antigen-binding domain” may include an antibody and its antigen-binding fragment. The term “antibody” is used herein in its broadest sense and encompasses a diverse range of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, complete immunoglobulins and their variants and fragments that are well known in the art and retain binding affinity to an antigen.

[0156] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting this population are identical except that they may have trace amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and target a single antigen epitope. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the antibody to be produced by some specific method. In some cases, monoclonal antibodies are produced by a single clone of B lymphocytes, or by cells or their offspring transfected with nucleic acids encoding the light and heavy chain variable regions of a single antibody (or its antigen-binding fragment). In some cases, monoclonal antibodies are isolated from the subject. Monoclonal antibodies may have conserved amino acid substitutions that do not substantially affect antigen-binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are well known, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New See York (2013).

[0157] Typically, immunoglobulins have heavy (H) and light (L) chains linked to each other by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). The major heavy chain has five classes (or isotypes), which determine the functional activity of antibody molecules (IgM, IgD, IgG, IgA, and IgE).

[0158] The heavy chain and light chain each contain a constant region (or constant domain) and a variable region (or variable domain) (for example, Kindt et al. Kuby Immunology, 6 th See ed., WH Freeman and Co., page 91 (2007). In some embodiments, heavy chain and The light chain variable regions combine to specifically bind to the antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camel antibodies consisting only of heavy chains are functional and stable even without the light chain (see, for example, Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). A reference to "VH" or "VH" refers to the variable region of the antibody heavy chain, including the variable region of the antigen-binding fragment, such as Fv, scFv, dsFv, or Fab. A reference to "VL" or "VL" refers to the variable domain of the antibody light chain, including those of Fv, scFv, dsFv, or Fab.

[0159] The variable regions of the light and heavy chains contain a "framework" region and three hypervariable regions (also called "complementarity-determining regions" or "CDRs") that interrupt it (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of different light or heavy chain framework regions are relatively conserved within the same species. The framework regions of the antibody, i.e., the framework regions of the constituent light and heavy chains, bind together to position and align the CDRs in three-dimensional space.

[0160] CDRs are primarily responsible for binding to antigen epitopes. The boundaries of a given CDR amino acid sequence are defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public). Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; The “Chothia” numbering scheme and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme) describe this. This can be easily determined using any of several well-known schemes, including the one described. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are further typically identified by the chain on which they are located. Thus, VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody containing it, and VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody containing it. Light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3.

[0161] "Antigen-binding fragments" are portions of full-length antibodies and diverse combinations of such portions that retain the ability to specifically recognize a congener antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments may be produced by modifying the entire antibody or newly synthesized using recombinant DNA methodologies. , containing antigen-binding fragments (for example, Kontermann and Dubel (Ed), Antibody Engineering, (See Vols. 1-2, 2nd Ed., Springer Press, 2010).

[0162] Single-chain antibodies (scFv) are genetically engineered molecules containing a gene fusion single-chain molecule formed by linking the VH and VL domains of one or more antibodies using a suitable polypeptide linker (e.g., Bird et al., Science, 242:423 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879 5883, 1988; Ahmad et al., Clin. Dev. See Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains within an scFv is typically not deterministic of the scFv. Therefore, scFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0163] In dsFv, the variable chains of the heavy and light chains have been stabilized by introducing disulfide bonds through mutation. Diabodies are also included, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain. However, because a linker that is too short to link the two domains into a single chain is used, the two domains are linked to complementary domains on another chain, forming two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444 6448, 1993; Poljak et al., Structure, 2:1121 1123, 1994).

[0164] Antibodies also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Chemical Co., Rockford, IL; Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0165] Antibodies that do not exist in nature can be constructed using solid-phase peptide synthesis, or produced by recombinant DNA, or, for example, Huse et al., Science 246:1275-1281 (1989) These can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains as described in (which are incorporated herein by reference). These methods, as well as other methods for producing, for example, chimeric, humanized, CDR grafts, single-chain, and bifunctional antibodies, are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of these is incorporated herein by reference).

[0166] A "reference antibody that binds to the same epitope" refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in an antagonist assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in an antagonist assay. Antibody antagonist assays are well known, and exemplary antagonist assays are provided herein.

[0167] A “humanized” antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (mouse, rat, or synthetic, etc.) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides this CDR is called the “donor,” and the human antibody or antigen-binding fragment that provides the framework is called the “acceptor.” In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. A constant region may or may not be present, but if present, it may be substantially identical to the human immunoglobulin constant region, for example, at least about 85-90% (about 95% or more, etc.). Thus, all parts of the humanized antibody or antigen-binding fragment (presumably (Excluding the CDR) is substantially identical to the corresponding portion of the natural human antibody sequence.

[0168] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies (typically from different species). In some examples, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0169] A “fully human antibody” or “human antibody” is an antibody that contains sequences from (or derived from) the human genome and does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions from (or derived from) the human genome. Human antibodies can be identified and isolated by using antibody production techniques based on human genome sequences, for example, by phage display or the use of genetically modified animals (see, for example, Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0170] An antibody may have one or more binding sites. If there is more than one binding site, these binding sites may be the same or different from each other. For example, natural immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and bispecific or bifunctional antibodies have two different binding sites.

[0171] Methods for testing the antibody ability to bind to any functional portion of a CAR are well known in the art, and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 Al, and U.S. Patent No. 7,338,929 below).

[0172] Furthermore, the CAR, the T cells expressing the CAR, the antibody, or its antigen-binding portion may include a detectable label such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), or an elemental particle (e.g., a gold particle).

[0173] C.conjugates DuoCAR, CAR-expressing T cells, or monoclonal antibodies or their antigen-binding fragments (all specific to one or more of the antigens disclosed herein) used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein may be conjugated to an agent such as an effector molecule or a detectable marker by any means of any of the many means known to those skilled in the art. Both covalent and non-covalent means may be used. The conjugate includes, but is not limited to, a molecule in which an antibody or antigen-binding fragment specifically bound to one or more of the antigens disclosed herein is covalently bound to an effector molecule or a detectable marker. Those skilled in the art will know of chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H, and 35 It is understood that a variety of effector molecules and detectable markers, including but not limited to radioactive agents such as S, as well as other labels, target sites, and ligands, can be used.

[0174] The selection of specific effector molecules or detectable markers depends on the specific target molecule or cell and the desired biological effect. Therefore, for example, effector —The molecule may also be a cytotoxin used to induce the death of specific target cells (such as tumor cells).

[0175] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody, resulting in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any of several well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). The linker may be any molecule used to bind the antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond between both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker may be bonded to the constituent amino acids via its side chains (e.g., to cysteine ​​via disulfide bonds) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acids.

[0176] In some embodiments, the linker may include a spacer element, which, when present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art, including U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521,28 Including Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (each of these in whole is incorporated herein by reference).

[0177] In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases an effector molecule or detectable marker from the antibody or antigen-binding fragment within the intracellular environment. In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleavage agent present within the intracellular environment (e.g., within lysosomes, endosomes, or caveoleas). The linker may be a peptide linker cleaved by a protease enzyme, including but not limited to intracellular peptidases, or lysosomal proteases or endosomal proteases. In some embodiments, the peptide linker is at least two amino acids long, or at least three amino acids long. However, the linker may be of a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, for example, 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids. The protease may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (e.g., Dubowchi See k and Walker, 1999, Pharm. Therapeutics 83:67-123. For example, thiol derivatives A peptide linker cleavable by the intracellular protease cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference. In one specific embodiment, the peptide linker cleavable by the intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0178] In another embodiment, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, such pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-unstable linkers that can be hydrolyzed within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, or ketals) can be used. (See, for example, U.S. Patents 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). These linkers are relatively stable under neutral pH conditions (such as in blood), but unstable at pH 5.5 or below 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (for example, a thioether bound to the therapeutic agent via an acylhydrazone linkage (see, for example, U.S. Patent No. 5,622,929)).

[0179] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are well known in the art, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), which can be formed using SPDB and SMPT. (e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., See also *In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer* (CW Vogel ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008). See also U.S. Patent No. 4,880,935.

[0180] In yet another specific embodiment, the linker is a malonate linker (Johnson et al. These are maleimide benzoyl linkers (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or 3'-N-amide analogs (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0181] In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released by antibody degradation (see U.S. Public Appeal No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0182] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), approximately 20%, 15%, 10%, 5%, 3%, or 1% of the linker in the conjugate sample is cleaved. Whether or not the linker is resistant to cleavage in the extracellular environment can be determined, for example, by examining the conjugate containing the linker of interest. The effect can be determined by incubating the compound with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of effector molecules or detectable markers released into the plasma. A variety of exemplary linkers that can be used in the conjugate are described in WO2004-010957, U.S. Public Notice No. 2006 / 0074008, U.S. Public Notice No. 20050238649, and U.S. Public Notice No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0183] In some embodiments, a conjugate is provided of a CAR, a CAR-expressing T cell, an antibody, or its antigen-binding moiety, and one or more small molecule toxins such as calicheamicin, meitansinoid, drastatin, auristatin, trichothecene, and CC1065, as well as derivatives of these toxins that have toxic activity.

[0184] Maytansin compounds suitable for use as maytansinoid toxin moieties are well known in the art, can be isolated from natural sources according to known methods, and can be produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or may Tansinol and maytansinol analogs can be prepared by synthesis according to well-known methods. Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic meitansinol and its derivatives and analogs are, for example, U.S. Patents No. 4,137,230, No. 4,248,870, No. 4,256,746, No. 4,260,608, No. 4,265,814, No. 4,294,757, No. 4,307,016, No. 4,308,268, No. 4,308,269, No. 4,309,428, No. 4, Conjugates containing maytansinoids, methods for preparing the same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated herein by reference. Conjugates containing maytansinoids, methods for preparing the same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent No. EP0425235 B1, the contents of which are expressly incorporated herein by reference.

[0185] Further toxins can be used in conjunction with CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties. Examples of toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. These toxins are well known in the art and many are readily available from commercial suppliers (e.g., Sigma Chemical Company, St. Louis, MO). The intended toxins also include variants of these toxins (see, for example, U.S. Patents 5,079,163 and 4,689,401).

[0186] Saporin is a toxin derived from Saponaria officinalis that inhibits protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin is not specific to cells. It lacks a mechanism for invading cells; therefore, in order to efficiently enter cells, it needs to bind to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein.

[0187] Diphtheria toxin is produced by Corynebacterium diphtheriae. They are separated. Typically, diphtheria toxins for use in immunotoxins have been mutated to reduce or eliminate nonspecific toxicity. The variant known as CRM107 has sufficient enzymatic activity but significantly reduced nonspecific toxicity and has been well known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and used in human clinical trials. See U.S. Patents No. 5,792,458 and 5,208,021.

[0188] Ricin is lectin RCA60 obtained from Ricinus communis (castor bean). See U.S. Patents 5,079,163 and 4,689,401 for examples of ricin. Ricinus communis agglutinin (RCA) exists in two forms, with molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120It is called (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). Chain A is responsible for inactivating protein synthesis and cell death. Chain B promotes the transport of chain A into the cytosol by binding lysine to galactose residues on the cell surface (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0189] Ribonucleases have also been used as immunotoxins by binding to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are described, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the engine antitumor antibiotic family that induces apoptosis by causing DNA double-strand breaks (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic site of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0190] Abrin contains toxic lectins derived from Abrus precatorius. Its toxic components, abrin a, b, c, and d, have a molecular weight of approximately 63–67 kD and consist of two polypeptide chains A and B linked by a disulfide bond. Chain A inhibits protein synthesis, while chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330–335, 1978).

[0191] CARs, CAR-expressing T cells, monoclonal antibodies, and their antigen-binding fragments (all specific to one or more of the antigens disclosed herein) used in patient-specific autologous antitumor lymphocyte cell populations may also be conjugated to detectable markers, such as those detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optics, and laparoscopy). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzymatic conjugation, radioisotypes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide luminescent agents. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also included. This is also useful. CAR, CAR-expressing T cells, antibodies, or their antigen-binding moieties may also be bound to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. If CAR, CAR-expressing T cells, antibodies, or their antigen-binding moieties are bound to a detectable enzyme, they can be detected by adding further reagents that, when used with the enzyme, produce a identifiable reaction product. For example, if horseradish peroxidase is present, a colored reaction product can be obtained by adding hydrogen peroxide and diaminobenzidine, which can be detected visually. CAR, CAR-expressing T cells, antibodies, or their antigen-binding moieties may also be bound to biotin and may be detected by indirectly measuring the binding of avidin or streptavidin. Notably, avidin itself may be bound to an enzyme or a fluorescent label.

[0192] CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties may be conjugated to paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be conjugated to lanthanides (such as europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may also be labeled with predetermined polypeptide epitopes that are recognized by a second reporter (such as a leucine zipper sequence pair, a secondary antibody binding site, a metal-binding domain, or an epitope tag).

[0193] CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties may also be conjugated with radiolabeled amino acids. Radiolabeling may be used for both diagnostic and therapeutic purposes. For example, radiolabeling may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabeling may be used therapeutically as a toxin to treat tumors in a target, for example, neuroblastoma. Examples of labeling for polypeptides are:3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 including, but not limited to, radioisotopes or radiolabeled nucleotides such as I.

[0194] Such means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, and fluorescent markers may be detected by detecting emitted light using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0195] D. Nucleotides, Expression, Vectors, and Host Cells According to one embodiment of the invention, there is further provided a nucleic acid comprising a nucleotide sequence encoding any of the DuoCARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants) described herein. The nucleic acids according to the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0196] In one embodiment, there is provided an isolated nucleic acid molecule encoding a chimeric antigen receptor (DuoCAR) comprising at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, in that order from the N-terminus to the C-terminus.

[0197] In one embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one single-strand variable fragment of an antibody that binds to the antigen.

[0198] In another embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to the antigen.

[0199] In yet another embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding the CAR is provided, wherein the encoded CAR extracellular antigen-binding domain comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to the antigen.

[0200] In one embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule is provided, in which the encoded extracellular antigen-binding domain is bound to a transmembrane domain by a linker domain.

[0201] In another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, where the encoded extracellular antigen-binding domain is located after a sequence encoding a leader peptide or signal peptide.

[0202] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, where the encoded extracellular antigen-binding domain targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0203] In a specific embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding the CAR is provided, wherein the encoded extracellular antigen-binding domains are: anti-CD19 scFV antigen-binding domain, anti-CD20 scFV antigen-binding domain, anti-CD22 scFV antigen-binding domain, anti-ROR1 scFV antigen-binding domain, anti-TSLPR scFV antigen-binding domain, anti-mesoterin scFV antigen-binding domain, anti-CD33 / IL3Ra scFV antigen-binding domain, anti-CD38 scFV antigen-binding domain, anti-CD123(IL3RA) scFV antigen-binding domain, anti-CD138 scFV antigen-binding domain, anti-BCMA(CD269) scFV antigen-binding domain, anti-GPC2 scFV antigen-binding domain, anti-GPC3 scFV antigen-binding domain, anti-FGFR4 scFV antigen-binding domain, anti-c-Met scFV antigen-binding domain, anti-PMSA scFV antigen-binding domain, and anti-glycolipid F77 The amino acid sequence comprising an scFV antigen-binding domain, an anti-EGFRvIII scFV antigen-binding domain, an anti-GD-2 scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these, or any combination thereof.

[0204] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, the DuoCAR provided herein further comprises a linker domain.

[0205] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular antigen-binding domain, an intracellular signaling domain, or both are linked to a transmembrane domain by a linker domain.

[0206] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, where the encoded linker domain is derived from the extracellular domain of CD8 and bound to the transmembrane domain.

[0207] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, isolated nucleic acid molecules encoding the CAR are provided, wherein the nucleic acid sequence encoding the transmembrane domain comprises a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0208] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded transmembrane domain comprises an amino acid sequence having at least one and not more than 10 modifications, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0209] In another embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain containing a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or combinations thereof.

[0210] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0211] In one embodiment of the CAR disclosed herein, an isolated nucleic acid molecule encoding the CAR is provided, in which the encoded intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

[0212] In another embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, wherein at least one intracellular signaling domain encoding comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0213] In a further embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, wherein at least one co-stimulatory domain encoding a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0214] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, further containing a leader sequence or a signal peptide sequence.

[0215] In some embodiments, the nucleotide sequence may be modified in terms of codons. While not bound by any theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve substituting native codons with other codons that encode the same amino acid but can be translated by tRNA that is more readily utilized in the cell, thus potentially increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that could interfere with translation, thus potentially increasing translation efficiency.

[0216] In one embodiment of the present invention, the nucleic acid may include a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the present invention, the nucleic acid may include a codon-modified nucleotide sequence encoding any of the DuoCARs (including their functional moieties and functional variants) described herein.

[0217] As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may be obtained from synthetic or natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide bonds (such as phosphoramide bonds or phosphorothioate bonds instead of phosphodiesters found between nucleotides in unmodified oligonucleotides). In some embodiments, the nucleic acid may contain no insertions, deletions, inversions, and / or substitutions at all. However, as described herein, in some examples, it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0218] Recombinant nucleic acids may have sequences that do not exist in nature, or sequences that artificially combine two distant regions within a sequence. These artificial combinations are often achieved by chemical synthesis, or more generally, by artificially manipulating distant nucleic acid regions using genetic engineering techniques, such as those described in the Sambrook et al. literature cited above. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic binding reactions using procedures well known in the art. See, for example, the Sambrook et al. and Ausubel et al. literature cited above. For example, nucleic acids may be chemically synthesized using natural nucleotides, or using nucleotides modified in various ways (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the double helix formed by hybridization. Examples of modified nucleotides that can be used for nucleic acid synthesis include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, and ino Syn, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methyl This includes, but is not limited to, toxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids according to the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0219] The nucleic acid may include any isolated or purified nucleotide sequence encoding any of the DuoCARs described above or its functional portion or functional variant. Alternatively, the nucleotide sequence may include a nucleotide sequence or combination of degenerate sequences degenerated into any of the sequences described above.

[0220] One embodiment further provides isolated or purified nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein, or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0221] Nucleotide sequences that hybridize under stringent conditions may hybridize under highly stringent conditions. “Highly stringent conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any nucleic acid described herein) in a detectably large amount compared to nonspecific hybridization. Highly stringent conditions include conditions that allow a polynucleotide with strictly complementary sequences or containing only 2-3 scattered mismatches to be distinguished from a random sequence that coincidentally contains 2-3 small regions (e.g., 3-10 bases) matching the nucleotide sequence. These small complementary regions are more readily dissolved than full-length complementary regions of 14-17 bases or more, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions are, for example, about 0.02-0.1M This may include low-salt and / or high-temperature conditions such as NaCl or equivalent at a temperature of approximately 50-70°C. Such highly stringent conditions allow for very little mismatch between the nucleotide sequence and the template or target chain, if any, and are particularly suitable for detecting the expression of any of the DuoCARs of the present invention. Generally, it is understood that the conditions can be made even stringier by increasing the amount of formamide added.

[0222] Also provided are nucleic acids comprising nucleotide sequences that are at least about 70% or more identical to any of the nucleic acids described herein, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0223] In one embodiment, nucleic acids may be incorporated into the recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids described above. For the purposes described herein, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct, wherein the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is brought into contact with a host cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the host cell. These are vectors that, when used in conjunction with host cells, enable the host cell to express the mRNA, protein, polypeptide, or peptide in question. Such vectors generally do not exist in nature.

[0224] However, some of these vectors may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, may be synthetic or partially obtained from natural sources, and may contain natural, unnatural, or mutated nucleotides. The recombinant expression vector may contain natural or unnatural nucleotide-nucleotide bonds, or both types of bonds. Preferably, unnatural or mutated nucleotides or nucleotide-nucleotide bonds do not interfere with the transcription or replication of the vector.

[0225] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector that can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for reproduction and proliferation, or for expression, or both (such as plasmids and viruses). The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0226] Bacteriophage vectors such as λυTIO, λυTI 1, λZapII (Stratagene), EMBL4, and λNM149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors or lentiviral vectors. Lentiviral vectors include, in particular, self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). These are vectors derived from at least a portion of the lentiviral genome. Other examples of lentiviral vectors that can be used in clinical practice include, but are not limited to, the LENTIVECTOR® gene transfer technology from Oxford BioMedica plc and the LENTIMAX® vector system from Lentigen, etc. Non-clinical lentiviral vectors are also available and may be well known to those skilled in the art.

[0227] Several transfection techniques are generally known in this field (e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al., supra; Davis). et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al, Gene, 13: 97 (1981)).

[0228] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al. above), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic acid transfer using a high-speed microparticle gun (see, e.g., Klein et al, Nature, 327: 70-73 (1987)).

[0229] In one embodiment, the recombinant expression vector may be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al. and Ausubel et al., as mentioned above. The circular or linear construct of the expression vector may be prepared to include a replication mechanism that functions in a prokaryotic or eukaryotic host cell. The replication mechanism may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, and bovine papillomavirus.

[0230] Recombinant expression vectors may include regulatory sequences such as transcription codons, translation start codons, and translation stop codons, which are appropriately specific to the type of host cell to which the vector is introduced (e.g., bacteria, fungi, plants, or animals), and which take into account whether the vector is DNA or RNA-based. Recombinant expression vectors may also include restriction sites to facilitate cloning.

[0231] Recombinant expression vectors may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include, for example, biocide resistance such as resistance to antibiotics and heavy metals, and nutritional complementation in the host to make it protrophic. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0232] Recombinant expression vectors may include a native or non-native promoter operably bound to a nucleotide sequence encoding a CAR (including its functional moiety and functional variants), or to a nucleotide sequence complementary to or hybridizing with the CAR-encoding nucleotide sequence. The choice of promoter (e.g., strong, weak, inducible, tissue-specific, and developmental-specific) is within the ordinary knowledge of those skilled in the art. Similarly, the binding of nucleotide sequences to promoters is also within the ordinary knowledge of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the terminal repeat sequence of a mouse stem cell virus.

[0233] Recombinant expression vectors may be designed for transient expression, stable expression, or both. Alternatively, recombinant expression vectors may be prepared for constitutive or inducible expression.

[0234] Furthermore, recombinant expression vectors may be constructed to include suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cell death in which it expresses a suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, to which the cell death occurs upon contact with or exposure to the agent. Suicide genes are well known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0235] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. Where used herein, the term “host cell” means “this.” This refers to any type of cell that may contain the recombinant expression vector according to the invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or it may be a prokaryotic cell, such as a bacterium or protist. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism, such as a human). The host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in a suspension). Suitable host cells are well known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. If the purpose is amplification or replication of the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. If the purpose is to produce a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, may originate from any type of tissue, and may be at any developmental stage, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0236] For the purposes described herein, T cells may be any T cells, including cultured T cells (e.g., primary T cells), T cells from cultured T cell lines (e.g., Jurkat, SupTl), or T cells obtained from mammals. If obtained from mammals, T cells may be obtained from a very diverse range of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells may be fortified or purified. T cells may be human T cells. T cells may be T cells isolated from humans. T cells may be any type of T cell, at any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Thi and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, and naive T cells. T cells may be CD8+ T cells or CD4+ T cells.

[0237] In one embodiment, the DuoCAR described herein can be used in preferred cells other than T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells derived from pluripotent stem cells.

[0238] Furthermore, one embodiment also provides a population of cells comprising at least one host cell as described herein. This population of cells may be a heterogeneous population comprising at least one other type of cell, for example, a host cell that does not contain any of the recombinant expression vectors described herein (e.g., a T cell), or a cell other than a T cell, for example, a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population mainly comprising (e.g., essentially consisting of) host cells comprising the recombinant expression vector. The population may also be a clonal cell population in which all cells of the population are clones of a single host cell comprising the recombinant expression vector, and therefore all cells of the population contain this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0239] DuoCAR (including its functional moiety and variants), nucleic acids, recombinant expression vectors, host cells (including its population), and antibodies (including its antigen-binding moiety) may be isolated and / or purified. For example, in a purified (or isolated) host cell preparation, the purity of the host cells is higher than that in the natural environment within the body. Such host cells may be prepared, for example, by standard purification techniques. In some embodiments, the host cell preparation is purified so that host cells constitute at least about 50%, for example, at least about 70%, of the total cell content of the preparation. For example, this purity is less Each may be approximately 50%, or may exceed approximately 60%, 70%, or 80%, or may be approximately 100%.

[0240] E. Treatment Method It is intended that DuoCAR, used in patient-specific autologous antitumor lymphocyte cell populations, may be used in methods for treating or preventing diseases in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in mammals, comprising the step of administering to a mammal DuoCAR, nucleic acid, recombinant expression vector, host cells, cell populations, antibodies and / or their antigen-binding moieties, and / or pharmaceutical compositions in amounts effective for treating or preventing cancer in mammals. Further uses of the DuoCAR described above are disclosed above.

[0241] One embodiment further includes a step of lymphocyte depletion in a mammal prior to administration of DuoCAR as disclosed herein. Examples of lymphocyte depletion include, but are not limited to, non-myeloablative lymphocyte depletion chemotherapy, myeloablative lymphocyte depletion chemotherapy, and total body irradiation.

[0242] For the purposes of this method, in which host cells or a population of cells are administered, these cells may be allogeneic cells of the mammal in question, or autologous cells of the mammal in question. Preferably, these cells may be autologous cells of the mammal in question. As used herein, "allogeneic" means any material that originates from an animal that is of the same species as the individual into which the material is introduced, but is a different individual. Two or more individuals are said to be allogeneic if they do not have identical genes at one or more loci. In some embodiments, allogeneic material from individuals of the same species may be genetically distinct enough to interact antigenically. As used herein, "autologous" means any material that originates from the same individual into which the material will later be introduced again.

[0243] As used herein, the mammal referred to may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals such as mice and hamsters and lagomorph mammals such as rabbits. The mammal may be a carnivore including the families Felidae (cat) and Canidae (dog). The mammal may be an artiodactyl including the subfamily Bovinae (cow) and swine (pig), or a perissodactyl including the family Equidae (horse). The mammal may be a primate, a ceboid, or a simoid (monkey), or an ape (human and monkey). Preferably, the mammal is a human.

[0244] With regard to the methods described above, the cancer may be any cancer including, but not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., urothelial carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer. Any cancer may be involved.

[0245] As used herein, the terms "treatment" and "prevention" and their derivatives do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that would be recognized by one of ordinary skill in the art as having the potential for benefit or a therapeutic effect. In this regard, the present method can provide any amount or any level of cancer treatment or prevention in a mammal.

[0246] Furthermore, the treatment or prevention provided by the present method may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented (e.g., cancer). Also, for the purposes described herein, "prevention" may include delaying the onset of a disease or its symptoms or conditions.

[0247] Another embodiment provides a method for detecting the presence of cancer in a mammal, which comprises: (a) forming a complex by contacting a sample comprising one or more cells from the mammal with DuoCAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition; and (b) detecting the complex, wherein detection of the complex suggests the presence of cancer in the mammal.

[0248] The sample may be obtained by any suitable method, such as a biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may be for the purpose of subjecting the removed tissue and / or cells to experimental methods, which may include experiments to determine whether the individual has a particular condition or disease state and / or is suffering therefrom. The condition or disease may be, for example, cancer.

[0249] In one embodiment of a method for detecting the presence of proliferative disorders in mammals, such as cancer, the sample containing mammalian cells may be a sample containing whole cells, their lysates, or whole cell lysates fractions, such as the nuclear or cytoplasmic fraction, the total protein fraction, or the nucleic acid fraction. If the sample contains whole cells, these cells may be any cells of a mammal, such as cells of any organ or tissue (including blood cells or endothelial cells).

[0250] The contact described above may occur in vitro or in vivo in mammals. Preferably, the contact occurs in vitro.

[0251] Furthermore, detection of the complex may be carried out by any of the multiple methods known in the art. For example, DuoCAR disclosed herein, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or antibodies, or their antigen-binding moieties, may be labeled with detectable labels such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles), as disclosed above.

[0252] Methods for testing the target cell recognition ability and antigen specificity of CARs are well known in the art. For example, Clay et al., J. Immunol, 163: 507-513 (1999) teach methods for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin-2 (IL-2)). In addition, the function of CARs may be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al, J. Immunol. 174: 4415-4423 (2005).

[0253] Another embodiment provides the use of DuoCAR, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or their antigen-binding moieties, and / or pharmaceutical compositions according to the present invention for treating or preventing proliferative disorders in mammals, such as cancer. The cancer may be any of the cancers described herein.

[0254] Any method of administration, including topical and systemic administration, may be used for the disclosed therapeutic agent. For example, administration may be used topically, orally, intravascularly (including intravenous), intramuscularly, intraperitoneally, intranasally, intradermally, subarachnoidally, and subcutaneously. The specific mode of administration and drug regimen may be selected by the attending clinician, taking into consideration the details of the case (e.g., the subject, the disease, the associated disease state, and whether the treatment is prophylactic). If more than one agent or composition is administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. The method of administration includes injection, in which the CAR, CAR T cells, conjugates, antibodies, antigen-binding fragments, or compositions are provided in a non-toxic and pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, non-volatile oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area after tumor removal or to an area suspected of having a tumor-like tendency. In some embodiments, sustained release of a pharmaceutical preparation containing a therapeutically effective amount of antibody or antigen-binding fragment into (or near) the tumor may be beneficial. In other examples, the conjugate may be applied topically as eye drops to the cornea or intravitreous tissue of the eye.

[0255] The disclosed therapeutic agents may be formulated in a unit dosing form suitable for administering precise doses one at a time. In addition, the disclosed therapeutic agents may be administered in single-dose or multi-dose schedules. A multi-dose schedule may involve more than one dose (e.g., 1 to 10 doses) administered individually in the initial series of treatments, followed by remaining doses at time intervals thereafter, as needed, to maintain or enhance the effects of the composition. Treatment may involve administering the compound once or multiple times a day (multi-daily doses) over a period of 2 to 3 days, several months, or even several years. Therefore, the drug regimen may be determined at least in part on the specific requirements of the subject being treated and may depend on the judgment of the administering physician.

[0256] Typical dosages of antibodies or conjugates may range from approximately 0.01 to approximately 30 mg / kg, for example, from approximately 0.1 to approximately 10 mg / kg.

[0257] In a specific example, the subject receives a therapeutic composition comprising one or more of the following: a conjugate, antibody, composition, DuoCAR, CAR T cells, or further agents, on a multiple daily dosing schedule, such as for at least two consecutive days and ten consecutive days, over a period of several weeks, several months, or several years. In one example, the subject receives the conjugate, antibody, composition, or further agent over a period of at least 30 days, for example, over a period of at least two months, at least four months, at least six months, at least twelve months, at least twenty-four months, or at least thirty-six months.

[0258] In some embodiments, the disclosed methods involve combining the disclosed antibody, antigen-binding fragment, conjugate, CAR, or T cells expressing the CAR (e.g., sequentially, substantially simultaneously, or concurrently) with surgery, radiotherapy, and / or chemotherapy. This includes providing such agents and treatments. Methods and therapeutic dosages for such agents and treatments are well known to those skilled in the art and may be determined by a skilled clinician. Preparations and administration schedules for further agents may be used in accordance with the manufacturer's instructions or at the discretion of a skilled physician based on experience. Preparations and administration schedules for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., MC Perry, Williams & Wilkins, Baltimore, Md.

[0259] In some embodiments, combination therapy may involve administering a therapeutically effective dose of an additional cancer inhibitor. Examples of additional therapeutic agents that can be used with combination therapy include, but are not limited to, microtubule-binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective doses) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. Methods and therapeutic dosages for such agents are well known to those skilled in the art and may be determined by a skilled clinician.

[0260] Further chemotherapeutic agents for combination immunotherapy include nitrogen mustards (e.g., chlorambutyl, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), alkylating agents such as busulfan, dacarbazine, mechloretamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and larcitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; and podophyllum. Plant alkaloids such as scutellaria (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), and vinca (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitemocyte antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and barurubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; and photosensitizers such as aminolevulinic acid, methyl aminolevulinate, sodium porfimer, and verteporfin.This also includes, but is not limited to, other agents such as alitretinoin, altretamine, amsacrin, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin difutitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masopropyl alcohol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and can be determined by a skilled clinician.

[0261] In certain embodiments of the present invention, cells activated and proliferated using the methods described herein or other methods known in the art for proliferating T cells to therapeutic levels are treated with any number of appropriate treatment modalities (antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or ephalisma treatment for psoriasis patients). The T cells according to the present invention are administered to the patient in conjunction with (for example, before, simultaneously with, or after) treatments using agents, including, but not limited to, mab treatment or other treatments for PML patients. In further embodiments, the T cells according to the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunosuppressants such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), or inhibit the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993). In a further embodiment, a cell composition according to the present invention It is administered to the patient in conjunction with (for example, before, simultaneously with, or after) bone marrow transplantation, T-cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition according to the present invention is administered after B-cell depletion therapy such as an agent that reacts with CD20 (e.g., rituxan). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In a particular embodiment, after transplantation, the subject receives an infusion of the proliferated immune cells according to the present invention. In an additional embodiment, the proliferated cells are administered before or after surgery.

[0262] The dosage of the above treatment administered to a patient may vary depending on the condition being treated and the specific characteristics of the patient receiving the treatment. Dosage determination in human administration is carried out as conventionally accepted. For example, the dosage of CAMPATH is generally in the range of 1 to approximately 100 mg for adult patients, usually administered once daily for 1 to 30 days. The preferred daily dose is 1 to 10 mg per day, but in some cases, higher doses such as 40 mg per day may be used.

[0263] Combination therapy can produce and demonstrate synergistic effects, meaning that the effect achieved when multiple active ingredients are used together is greater than the combined effect obtained when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit dose preparation, (2) delivered alternately or in parallel as separate preparations, or (3) used in conjunction with some other regimen. In the case of alternate delivery, synergistic effects can occur when the compounds are administered or delivered sequentially, for example, by injection separately using separate syringes. Generally, in the case of alternate delivery, the effective dose of each active ingredient is administered sequentially, i.e., consecutively, whereas in combination therapy, the effective doses of two or more active ingredients are administered together.

[0264] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anticancer treatment. After a sufficient amount of time has elapsed, the administered antibody or antigen-binding fragment or conjugate forms an immune complex with the antigen expressed on each cancer cell, and this immune complex is then detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained before the treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control obtained before the treatment indicates that the treatment is effective.

[0265] F. Biopharmaceutical Composition A biopharmaceutical composition or biological composition (hereinafter, "composition") containing one or more of the disclosed DuoCARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates, DuoCARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein in a carrier (such as a pharmaceutically acceptable carrier) is provided herein for use in gene therapy, immunotherapy, adoptive immunotherapy, and / or cell therapy. This composition may be prepared in unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. This composition may be formulated for systemic (such as intravenous) or local (such as intratumoral) administration. In one example, the disclosed DuoCARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration such as intravenous administration. A composition containing a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment disclosed herein is useful, for example, in the treatment and detection of tumors (such as, but not limited to, neuroblastoma). In some examples, this composition is useful in the treatment or detection of cancer. A composition containing a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment disclosed herein is also useful, for example, in the detection of pathological angiogenesis.

[0266] This administration composition may contain a solution in which CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, is dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such solutions are sterile and generally free of undesirable substances. The composition may be sterilized by conventionally known sterilization techniques. To approximate physiological conditions, the composition may contain, as necessary, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates in the preparation may vary over a wide range and may be selected according to the requirements of a particular administration mode and target, mainly based on the volume, viscosity, and weight of the fluid. Practical methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are well known or will be apparent to those skilled in the art.

[0267] A typical composition for intravenous administration contains approximately 0.01 to 30 mg / kg per subject per day of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or a conjugate containing CAR-expressing T cells, antibody or antigen-binding fragment). The actual preparation methods of the administration composition may be well known or obvious to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0268] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be provided in lyophilized form and administered after reconstitution with sterile water, or they may be provided dissolved in sterile solutions of known concentrations. The DuoCAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates solution, is then filled into an infusion bag containing 0.9% sodium chloride (USP) and administered, in some cases, at a dosage of 0.5–15 mg / kg body weight. There is considerable experience in the art with the administration of antibody or antigen-binding fragments and conjugate drugs; for example, antibody drugs have been available on the US market since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, their antigen-binding fragments, and conjugates, may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher load dose is administered, followed by a lower maintenance dose. For example, if the antibody or antigen-binding fragment is an initial loading dose of 4 mg / kg (or antibody or antigen The conjugate containing the binding fragment (the corresponding dose) is infused over approximately 90 minutes. If this initial dose is well tolerated, a maintenance dose of 2 mg / kg may then be administered weekly for 30 minutes each time for 4 to 8 weeks.

[0269] Controlled-release parenteral preparations may be prepared as grafts, oily injections, or particle systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central nucleus. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than approximately 1 μm, microspheres, and microcapsules are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries are approximately 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0270] Polymers may be used for the ion-controlled release of DuoCARs, or compositions of CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous, mobile liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for preparations and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite may be used for the controlled release of proteins. It is used as a microcarrier for release (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release of lipid-encapsulated drugs. And used for drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, therapeutic A great many systems are known for the controlled delivery of ferroproteins (US Patent Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735, 5,019,369, 5,055,303, 5,514,670, 5,413,797, 5,268,164, 5,004,697, 4,902,505, 5,506,206, 5,271,961, 5,254,342, and 5,534,496).

[0271] G. Kitt In one embodiment, kits using the DuoCARs disclosed herein are further provided. For example, kits for treating tumors in a subject or for producing CAR T cells expressing one or more of the DuoCARs disclosed herein. Such kits may typically include antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, DuoCARs, or T cells expressing CARs may be included in the kit.

[0272] The kit may include a container and a label or accompanying information attached to or accompanying the container. Preferred containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Typically, the container contains a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, DuoCARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured with a subcutaneous needle). The label or accompanying information indicates that the composition is to be used for the treatment of a particular condition.

[0273] The label or accompanying information may typically further include a description of the use of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, DuoCAR, or CAR-expressing T cells in, for example, a method of treating or prophylactically treating a tumor or a method of producing CAR T cells. The accompanying information typically includes instructions that are customarily included in the market packaging of a therapeutic product, which include information on indications, usage, dosage, administration, contraindications, and / or warnings related to the use of the therapeutic product. The contents of the instructions may be written in electronic format (e.g., floppy disk or compact disk) or visual format (e.g., video file). The kit may further include additional components to advance the specific use for which the kit was designed. For example, the kit may further include labeling detection means (e.g., enzyme substrates for enzyme labeling, filter sets for fluorescent labeling detection, or appropriate secondary labels such as secondary antibodies). The kit may further include buffers and other reagents that are customarily used in carrying out a particular method. Such kits and appropriate contents are well known to those skilled in the art. [Examples]

[0274] Examples The present invention is further illustrated by the embodiments of DuoCAR shown in the accompanying drawings and on pages 17-27 of this disclosure (including those mentioned above), but these embodiments should not be construed as limiting the scope of the invention. On the contrary, it will be clearly understood that various other embodiments, modifications, and equivalents will be necessary, and such other embodiments, modifications, and equivalents will come to mind for those skilled in the art after reading this specification, without departing from the spirit of the invention and / or the scope of the accompanying claims.

[0275] Various details are described in conjunction with the exemplary implementations outlined above, but we believe that various alternatives, modifications, alterations, improvements, and / or substantial equivalents (whether known or not, or whether currently foreseeable or foreseeable) will become apparent by referring to the disclosures set forth above.

[0276] Each application and patent cited herein, as well as each document or prior art document cited within each application and patent (including each issued patent in litigation, “Application Reference”), as well as each PCT application or patent and foreign application or patent corresponding to and / or claiming priority over any of the said applications and patents, and each document cited or referenced within each Application Reference, are expressly incorporated herein by reference and may be used in practice of the present invention. More generally, a document or prior art document is cited within the text, in the prior art document list prior to the claims, or within the text itself, and each of such documents or prior art documents (“Prior Art Document Cited herein”), and each document or prior art document cited within each Prior Art Document Cited herein (including manufacturer’s standards, instructions, etc.) are expressly incorporated herein by reference.

[0277] The above-described examples of several specific embodiments are intended to allow others, by applying the latest knowledge, to adapt these specific embodiments for diverse uses without deviating from the general concept. This specification provides sufficient information to easily modify or adapt, and such adaptations and modifications should therefore be understood to be within the meaning and scope of the equivalent of the disclosed embodiment. It should be understood that the words or technical terms used herein are for illustrative purposes only and not for limiting purposes. Exemplary embodiments are disclosed in the drawings and descriptions, and certain terms may be used, but unless otherwise noted, these are for general and illustrative purposes only and not for limiting purposes, and therefore do not limit the scope of the claims. Furthermore, it should be understood that certain steps of the methods disclosed herein may be performed in a different order or in a combination of steps. Accordingly, it is intended that the claims accompanying the specific embodiments disclosed herein are not limited. Many equivalents of the embodiments of the present invention described herein can be recognized by those skilled in the art, or grasped by conventional experimental methods alone. Such equivalents are included in the following claims.

[0278] Description of Examples Four examples are presented, demonstrating the feasibility of the Duo set technology by showing that combinations of three functional binding domains and different co-stimulatory intracellular domains are expressed on the surface of LV-transfected human T cell populations (Example 1), and demonstrating its effectiveness by showing that the population has functional activity against three different leukemia antigens (Example 2). A comparison of the expression and function of DuoCARs produced by simultaneous transfection, i.e., transfection with a single LV product encoding both DuoCAR chains (created by simultaneously transfecting a packaging strain with two plasmids encoding CARs), is described in Example 3. In Example 4, the transfection efficiency and function of DuoCARs transfected with LVs produced by the simultaneous transfection method are compared with bicistronic DuoCARs encoded by a single construct (with a ribosome skipping site between the two DuoCAR chains).

[0279] An example of a single-specific CAR (which forms the basis of this technology and can be included as a Duo set element in DuoCAR) is the single CD20-targeting vector LTG1495 (nucleotide sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4). A second example is the CD22-specific single-specific CAR LTG2200 (nucleotide sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10). Important molecular aspects in DuoCAR construction include including non-repeating compatible sequences and evaluating these sequences in transduced T cells to prevent inappropriate recombination or intracellular binding. These can occur in both the vector-producing cell line and the target cell population. For this reason, variant CAR structures known to be compatible with the DuoCAR environment are included. These include the CD19-specific CAR LTG1494 (nucleotide sequence: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30). This sequence contains a linker known as the Whitlow linker (amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 107), see Whitlow M., et al., 1993, Protein Eng. 6:989-995), which has been clearly described as joining the heavy and light chains of scFv. In some cases, the Whitlow linker is (GGGGS) n A linker was substituted (for example, the CD19 CAR form in LTG1538 (nucleotide sequence is SEQ ID NO: 31, amino acid sequence is SEQ ID NO: 32)). In another example, a CAR with a different transmembrane domain was constructed. Anti-CD19 CAR LTG1562 (nucleotide sequence SEQ ID NO: 21, amino acid sequence SEQ ID NO: 22) utilizes the CD4 (not CD8) transmembrane domain. Similarly, anti-CD19 CAR LTG1563 has another transmembrane form derived from TNFRSF19 (nucleotide sequence SEQ ID NO: 49, amino acid sequence SEQ ID NO: 50). DuoCARs can also target solid tumors (e.g., those expressing mesothelin tumor antigen). For example, an scFV binder for mesothelin has been developed (concurrently pending provisional patent application No. 62 / 444,201 by the same applicant (title of invention: "Treatment of cancer by anti-mesoselin immunotherapy"). A composition and method for doing so is disclosed in application filed on 9 January 2017 (Lentigen Technology, case number LEN_017); the nucleotide sequence is SEQ ID NO: 37, the amino acid sequence is SEQ ID NO: 38), which can be incorporated into a functional CAR (nucleotide sequence is SEQ ID NO: 39, the amino acid sequence is SEQ ID NO: 40), and thus can be incorporated into DuoCAR therapy. In addition to the scFv sequence, a single-stranded antigen binder (not scFv) can also be incorporated into DuoCAR applications. For example, a CD33-specific heavy chain-only binder (disclosed in the concurrently pending provisional patent application No. 62 / 476,438 by the same applicant (title of invention "Composition and Method for Treating Cancer by Anti-CD33 Immunotherapy," filed March 24, 2017, Lentigen Technology, case number LEN_018); nucleotide sequence is SEQ ID NO: 41, amino acid sequence is SEQ ID NO: 42) can be incorporated into the functional CAR LTG1906 (nucleotide sequence is SEQ ID NO: 43, amino acid sequence is SEQ ID NO: 44) to target CD33-expressing malignancies. One example of a DuoCAR therapeutic application is the treatment of leukemia expressing CD19, CD20, and TSLPR antigens. In this case, LTG1496 or LTG We were able to combine 1497 (sequence numbers 35 and 26, respectively) with a TSLPR-specific CAR (LTG1789) (sequence number 47, and amino acid sequence number 48) created from a TSLPR-specific scFV domain (nucleotide sequence number 45, amino acid sequence number 46).

[0280] An example of a tandem CAR (containing two scFv domains, nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24) that forms the basis of this technology is CD20_CD19 CAR LTG1497 (nucleotide sequence SEQ ID NO: 25, amino acid sequence SEQ ID NO: 26). In some cases, reversing the order of these two binders can improve DuoCAR expression in target cells. Therefore, both LTG1497 (nucleotide sequence SEQ ID NO: 25, amino acid sequence SEQ ID NO: 26), in which the CD19 scFv is relatively close to the membrane, and LTG1496 (nucleotide sequence SEQ ID NO: 33, amino acid sequence SEQ ID NO: 34), in which the CD19 scFv is relatively farther away, can be used as components of a Duo set containing a DuoCAR.

[0281] Methods used in Examples 1 and 2: Cell lines (PBMCs and targets) All cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) unless otherwise noted. Burkitt lymphoma cell line Raji, acute lymphoblastic leukemia cell line REH, and chronic myeloid leukemia cell line K562 were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). Human embryonic kidney cell line 293T was grown in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated FBS.

[0282] Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor cells with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD), followed by cloning and selecting luciferase-positive clones. The Raji clone, which stably expresses firefly luciferase, was then used to create NSG mice (NOD.Cg-Prkd scid Il2rg tm1Wjl Mouse-compatible Raji-luc strains were created by transplanting them into (SzJ, The Jackson Laboratory, Sacramento, CA) cells, and then positively selected (CD19 microbeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negatively selected (mouse cell depletion kit, Miltenyi Biotec) cells. Transplanted Raji-luc tumor cells were isolated from mouse spleens by either of the following methods, cultured and grown, and then re-cloned to facilitate the selection of high-expression clones of firefly luciferase. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI, Oklahoma City, OK) with written consent from the donors. Processed buffy coat was purchased from OBI. CD4-positive and CD8-positive human T cells were purified from the buffy coat by positive selection using a 1:1 mixture of CD4 microbeads and CD8 microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0283] Production of chimeric antigen receptor (CAR) expression vectors including DuoCAR The sequences of the CAR antigen-binding domain scFv were derived from mouse hybridoma FMC-63 (FMC-63:AA 1-267, GenBank ID:HM852952.1) for CD19 and from Leu-16 for CD20 [1] (entire sequences of VL and VH). CD22 scFv binding was constructed from commonly available sequences. Tandem CAR19_20 or CAR20_19 were constructed by binding the scFv of each antibody in frame to the CD8 hinge / transmembrane domain (AA 123-191, reference sequence number NP_001759.3), the 4-1BB (CD137, AA 214-255, UniProt sequence number Q07011) transactivation domain, and the CD3 zeta signaling domain (CD247, AA 52-163, reference sequence number NP_000725.1). The scFv regions at 19A and 20A were sequentially linked by the mobile interchain linker (GGGGS) 5 (SEQ ID NO: 108), followed by the linkage of the CD8, 4-1BB, and CD3 zeta domains. As described in [2], a leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs. The sequences of the CAR constructs were codon-optimized (DNA2.0, Newark, CA) and cloned into a third-generation lentiviral plasmid backbone under the control of the human EF-1α promoter (Lentigen Technology, Gaithersburg, MD). As previously described in [3], the supernatant containing the lentiviral vector (LV) was obtained by transient transfection of HEK 293T cells. The lentiviral supernatant was collected in pellet form and stored at -80°C.

[0284] Transduction of primary T cells: Selected CD4+ and CD8+ human primary T cells from normal donors were raised in TexMACS medium (serum-free) supplemented with 40 IU / ml of IL-2, resulting in a cell density of 0.3–2 × 10⁶. 6The cells were cultured at a concentration of cells / ml and activated using CD3 / CD28 MACS® GMP TransAct reagent (Miltenyi Biotec). On day 3, transduction was performed overnight using a lentiviral vector encoding the CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO). The culture medium was changed on day 4. On day 5, the culture was transferred to TexMACS medium supplemented with 200 IU / ml IL-2 for growth and harvested on days 10-13.

[0285] Immunotherapy Effector Assay: To investigate cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were incubated overnight with CAR T cells in various effector target ratios. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was analyzed using an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). The assay range was determined using wells containing only targets (maximum CPS) and wells containing only targets with 1% Tween-20 added (minimum CPS). Specific lysis ratio The sum was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)).

[0286] Flow cytometry analysis: Unless otherwise noted, all cell staining reagents used for flow cytometry were obtained from Miltenyi Biotec. One million transduced CAR T cells were harvested from the culture, washed twice in chilled staining buffer (0.5% bovine serum albumin-supplemented AutoMACS solution), and pelletized at 4°C at 350×g for 5 minutes. First, CAR surface expression of transduced T cells was detected by staining with protein L-biotin conjugate (storage solution: 1 mg / ml, 1:1000 dilution, GenScript, Piscataway, NJ) at 4°C for 30 minutes. Subsequently, the cells were washed twice and stained with streptavidin-PE conjugate (storage solution: 1.0 ml, 1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) at 4°C for 30 minutes. Untransduced and transduced cells (stained with streptavidin-PE only) were used as negative controls. Anti-CD4 antibody was added during the second incubation step to determine the CD4 to CD8 ratio in the CAR T-positive population. Dead cells were removed by 7AAD staining (BD Biosciences, San Jose, CA). After washing the cells twice and resuspending them in 200 μl of staining buffer, quantitative analysis was performed by flow cytometry. Human T cells activated with CD3-CD28 nanomatrix (TransAct, Miltenyi Biotec) and transduced with a Duo-set vector in the presence of IL-2 were stained with specific Duo-set CAR T staining, and the expression of the CD19-, CD20-, or CD22-scFv domains of the antibodies was analyzed by flow cytometry using recombinant CD19, CD20, or CD22 for staining.

[0287] Anti-CD19 scFv activity was detected using CD19-Fc (R&D Biosystems) at 1 μg / sample, and stained with goat anti-human Fc-gamma-R-PE (Jackson ImmuoResearch Laboratories) at 0.75 μg / sample. Anti-CD20 scFv activity was detected using CD20-biotin (Miltenyi Biotech) at 0.1 μg / sample, and streptavidin pAPC (Miltenyi Biotec) at 0.2 μg / sample. Anti-CD22 scFc activity was detected using 0.1 μg / sample of D22-His (Thermo Fisher) and anti-His FITC (Miltenyi Biotec). Flow cytometry analysis was performed using MACSQuant® 10 Analyzer (Miltenyi Biotec). Target tumor lines and luciferase-positive subclones were characterized using CD19-FITC, CD20 VioBlue, and CD22-APC antibodies. Dead cells were removed from the analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0288] Example 1 Expression of DuoCAR (2+1 Duo set) in primary human T cells As proof of principle, a Duo set containing two CAR-T vectors was constructed. One component of this set expressed a tandem CD20_CD19 binding domain (LTG2228) (SEQ ID NOs. 51 and 52) bound to the CD8 transmembrane domain and the CD28 and CD3-zeta signaling domains. The second component of the Duo set was a CAR construct (LTG2200) (SEQ ID NOs. 9 and 10) in which one CD22 binder was bound to the CD8 transmembrane domain and the 4-1BB and CD3-zeta signaling domains. In Figure 7, in the paired columns, the left column shows double staining of CD20 scFv and CD19 scFv, and the right column shows double staining of CD22 scFv and CD19 scFv. The first row shows untransduced T cells (UTDs). Thus, it is shown that there is no binding. The second row shows T cells transduced with LV encoding the CD20_CD19 CAR vector (20-19-28z), which has a CD8 transmembrane domain and intracellular CD28 and CD3-zeta signaling domains. Double staining for CD20 and CD19 binding is seen (left panel), but only CD19 binding is seen in the right panel. The third row shows T cells transduced with the CD22 CAR vector (22-BBz), which has a CD8 transmembrane domain and intracellular 4-1BB and CD3-zeta signaling domains. No double staining for CD19 or CD20 is seen (left panel), indicating that only one cell population was able to bind to CD22 (right panel). In the fourth row, T cells are transduced with a Duo set containing both the second and third vectors. Only this Duo set expresses all three CAR coding binding domains (42% of cells express CD20_19 (left panel), and 38% express CD22 and CD19 binding domains (right panel)). Since CD22 and CD19 scFv are present in each of the two distinct transmembrane proteins containing the Duo set, the 38% represents a population that truly expresses the Duo set in this example.

[0289] Example 2 Antileukemia activity of human T cell preparations expressing DuoCAR, produced by simultaneous transduction. Antileukemic activity of human T cell preparations expressing DuoCAR (simultaneously targeting three leukemic antigens) (see Figure 7 for characteristics of DuoCAR expression). A Duo set containing a CD20_19 tandem CAR and a CD22-specific single CAR (prepared as in Example 1) was used as the effector T cell population in a cytotoxic T cell assay (using leukemic cell lines and model cell lines as targets). Human T cells transduced with a single CAR element (20_19-28z or 22-BBz) or DuoCAR (20_19-28z + 22-BBz) were used in a cytotoxic T cell assay at four different effector target ratios (20:1, 10:1, 5:1, and 2.5:1 as shown) (see Figure 8). The leukemia cell lines used as CAR-T targets were: Raji (expressing all three target antigens), REH (expressing all three target antigens), K562 (control, no target expression), K562-CD19 (expressing CD19), K562-CD20 (expressing CD20), and K562-CD22 (expressing CD22). Only DuoCAR transduced cells (20-19-28z + 22-BBz) showed high cytolytic activity against both leukemia cell lines (Raji and REH) and all three single-expression K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). This demonstrates that DuoCAR technology can uniquely and simultaneously target three leukemia antigens within the same effector T cell population, and therefore possesses superior antineoplastic activity because it can target one or more target antigens at once, thereby suppressing the development of escape variants (which cause malignant lesions) (cell clones that escape immunodeficiency due to the loss or downregulation of one or two antigens). Ultimately, it is thought that patient cure rates will increase as antigen deletion variants escape and proliferate (ultimately leading to relapse).

[0290] Example 3 Antileukemia activity of human T cell preparations expressing DuoCAR, produced by simultaneous transfection. The DuoCAR technology described herein generates a population of therapeutic lymphocytes (in this example, human T cells) expressing more than two antigen specificities from more than one transmembrane protein encoded by a gene vector. In this example, this is carried out by two different means. Figure 9 includes three rows of data labeled “No Transduction,” “Simultaneous Transduction,” and “Simultaneous Transfection.” Figure 9 includes two columns of data prepared in Figure 7, the first column showing the expression of CD20 and CD19-specific binding analyzed by flow cytometry, and the second column showing the expression of CD22 and CD19-binding activity analyzed by flow cytometry. The data was analyzed using tometry. The first row shows untransduced human T cells. No binding activity was observed for CAR-derived binding activity indicators such as CD19, CD20, or CD22 recombinant proteins, indicating that DuoCAR was not expressed. In the second row, DuoCAR was generated using "simultaneous transduction." In this dataset, two LVs were used to simultaneously transduce activated T cells. As shown in Figure 7, in the Duo set containing DuoCAR, one CAR was a tandem CD20 and CD19 binder bound to the CD28 signaling motif and the CD3-zeta signaling motif, while the other CAR was a CD22 binder bound to the 4-1BB and CD3-zeta signaling motif. The upper right quadrant of the first column shows a very distinctive pattern of single-staining of CD20 and CD19-scFv activity. This is because both binders are located on the same surface glycoprotein and are therefore co-expressed at equal intensity, thus producing a very distinctive linear pattern. The second column of the simultaneous transduction data shows a relatively conventional pattern, with the expression patterns of the two glycoproteins on each cell being unequal. Thus, four different population patterns are observed. The lower left quadrant shows cells that do not express either binder. The upper left shows cells that express only the CD22 CAR. The lower right quadrant shows cells that express only the CD20_CD19 tandem CAR. Finally, the upper right quadrant shows cells that express both components of the CAR Duo set, including the DuoCAR.

[0291] In the bottom row, a cell population expressing DuoCAR is generated using a different method. In the simultaneous transduction method, two independently prepared LV preparations are used for T cell transduction, but "simultaneous transfection" refers to a method of producing LV by simultaneously transfecting a 293T packaging cell line with two backbone plasmids (encoding two CARs, including DuoCAR). The helper plasmid containing this third-generation LV system is the same in both methods. The advantage of the simultaneous transfection method is that a single LV preparation containing vectors encoding both CARs is produced. As can be seen from the data, when using the simultaneous transfection method, the expression patterns of CD20-CD19 CAR and CD22 CAR are almost the same as in the simultaneous transduction method in the second row. The staining patterns of both glycoproteins induced by LV produced by simultaneous transfection in the upper right quadrant of the second column of data (CD22 co-staining for CD22-CAR and CD19 co-staining for CD20_19 CAR) demonstrate that DuoCARs can be efficiently produced by either method.

[0292] References: 1)Wu, AM, et al., Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein engineering, 2001. 14(12): p. 1025-1033. 2) Haso, W., et al., Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood, 2013. 121(7): p. 1165-1174. 3)Kuroda, H., et al., Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection. Journal of virological methods, 2009. 157(2): p. 113-121. Example 4 DuoCAR and Bi-Cistronic Duo fabricated by simultaneous transfection method Comparison with CAR constructs Method used in Example 4: Cell lines (PBMCs and targets): Unless otherwise noted, all cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). Burkitt lymphoma cell line Raji, acute lymphoblastic leukemia cell line REH, and chronic myeloid leukemia cell line K562 were used in 10% heat-inactivated IVF. Human embryonic kidney cell line 293T was cultured in RPMI-1640 medium supplemented with fetal serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). Human embryonic kidney cell line 293T was grown in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated FBS.

[0293] Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor cells with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD), followed by cloning and selecting luciferase-positive clones. The Raji clone, which stably expresses firefly luciferase, was then used to create NSG mice (NOD.Cg-Prkd cscid Il2rg tm1WjlMouse-compatible Raji-luc strains were created by transplantation (SzJ, The Jackson Laboratory, Sacramento, CA). Transplanted Raji-luc tumor cells were isolated from mouse spleens by either positive selection (CD19 microbeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negative selection (mouse cell depletion kit, Miltenyi Biotec), cultured, and re-cloned to facilitate the selection of high-expression clones of firefly luciferase. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI, Oklahoma City, OK) with written consent from the donors. Processed buffy coat was purchased from OBI. From the buffy coat, CD4-positive and CD8-positive human T cells were purified by positive selection using a 1:1 mixture of CD4 microbeads and CD8 microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0294] Construction of chimeric antigen receptor (CAR) expression vectors including DuoCAR: The sequences of the CAR antigen-binding domain scFv were derived from mouse hybridoma FMC-63 (FMC-63:AA 1-267, GenBank ID:HM852952.1) for CD19 and from Leu-16 for CD20 [1] (entire sequences of VL and VH). Several anti-CD22 scFv binding sequences were used. Tandem CAR19_20 or CAR20_19 were constructed by binding the scFv of each antibody in frame to the CD8 hinge-transmembrane domain (AA 123-191, PICS ID NP_001759.3), the 4-1BB (CD137, AA 214-255, UniProt SEQ ID Q07011) transactivation domain, and the CD3 zeta signaling domain (CD247, AA 52-163, PICS ID NP_000725.1). The scFv regions at 19A and 20A were sequentially linked by the mobile interchain linker (GGGGS) 5 (SEQ ID NO: 108), followed by the linkage of the CD8, 4-1BB, and CD3 zeta domains. As described in [2], a leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs. In the bicistronic CAR design, the two CAR strands are encoded within the same expression cassette, separated by the ribosome skip element 2A. The sequences of the CAR constructs were codon-optimized (DNA2.0, Newark, CA) and cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology, Gaithersburg, MD) under the control of the human EF-1α MSCV promoter. As previously described in [3], HEK Lentiviral vector (LV) supernatant was obtained by transient transfection of 293T cells. For simultaneous transfection experiments, equal amounts of two transfer plasmids encoding each DuoCAR strand were combined with a helper plasmid and given to the HEK 293T packaging cell line during the transfection process. The resulting viral vector preparation was used for transduction of primary human T cells. The pelletized lentiviral supernatant was collected and stored at -80°C.

[0295] Primary T cell transduction: Selected CD4+ and CD8+ human primary T cells from normal donors were transduced in TexMACS medium (serum-free) supplemented with 40 IU / ml of IL-2, resulting in a cell density of 0.3–2 × 10⁶. 6 The cells were cultured at a concentration of cells / ml and activated using CD3 / CD28 MACS® GMP TransAct reagent (Miltenyi Biotec). On day 3, transduction was performed overnight using a lentiviral vector encoding the CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO). The culture medium was changed on day 4. On day 5, the culture was transferred to TexMACS medium supplemented with 200 IU / ml IL-2 for growth and harvested on days 10-13.

[0296] Immunotherapy Effector Assay: To investigate cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were incubated overnight with CAR T cells in various effector target ratios. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was analyzed using an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). The assay range was determined using wells containing only targets (maximum CPS) and wells containing only targets with 1% Tween-20 added (minimum CPS). The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)).

[0297] Flow cytometry analysis: Unless otherwise noted, all cell staining reagents used for flow cytometry were obtained from Miltenyi Biotec. One million transduced CAR T cells were harvested from the culture, washed twice in chilled staining buffer (0.5% bovine serum albumin-supplemented AutoMACS solution), and pelletized at 4°C at 350×g for 5 minutes. First, CAR surface expression of transduced T cells was detected by staining with protein L-biotin conjugate (storage solution: 1 mg / ml, 1:1000 dilution, GenScript, Piscataway, NJ) at 4°C for 30 minutes. Subsequently, the cells were washed twice and stained with streptavidin-PE conjugate (storage solution: 1.0 ml, 1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) at 4°C for 30 minutes. Untransduced and transduced cells (stained with streptavidin-PE only) were used as negative controls. Anti-CD4 antibody was added during the second incubation step to determine the CD4 to CD8 ratio in the CAR T-positive population. Dead cells were removed by 7AAD staining (BD Biosciences, San Jose, CA). After washing the cells twice and resuspending them in 200 μl of staining buffer, quantitative analysis was performed by flow cytometry. Human T cells activated with CD3-CD28 nanomatrix (TransAct, Miltenyi Biotec) and transduced with a Duo-set vector in the presence of IL-2 were stained with specific Duo-set CAR T staining, and the expression of the CD19-, CD20-, or CD22-scFv domains of the antibodies was analyzed by flow cytometry using recombinant CD19, CD20, or CD22 for staining.

[0298] Anti-CD19 scFv activity was detected using CD19-Fc (R&D Biosystems) at 1 μg / sample, and stained with goat anti-human Fc-gamma-R-PE (Jackson ImmuoResearch Laboratories) at 0.75 μg / sample. Anti-CD20 scFv activity was detected using CD20-biotin (Miltenyi Biotech) at 0.1 μg / sample, and streptavidin APC (Miltenyi Biotec) at 0.2 μg / sample. Anti-CD22 scFv activity was detected using 0.1 μg / sample of D22-His (Thermo Fisher) and anti-His FITC (Miltenyi Biotec). Flow cytometry analysis was performed using MACSQuant® 10 Analyzer (Miltenyi Biotec). Target tumor lines and luciferase-positive subclones were characterized using CD19-FITC, CD20 VioBlue, and CD22-APC antibodies. Dead cells were removed from the analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0299] Construction of a bicistronic DuoCAR using a 2A ribosome skipping sequence In addition to the simultaneous transfection and simultaneous introduction approach described in Examples 2 and 3 above (DuoCAR, which simultaneously targets three hematological tumor antigens CD19, CD20, and CD22 and has different costimulatory domains), the use of the self-cleaving element 2A can promote the simultaneous expression of two CAR chains from a single mRNA transcript. The 2A element intervenes in ribosome skipping when the mRNA transcript is translated into protein, thereby enabling the production of two separate CAR protein chains in equimolar ratios. In this example, the first CAR chain consists of a CD22 scFv bound in-frame to a CD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta-activating domain. The second CAR chain consists of a targeting domain based on tandem CD20 CD19 scFv, followed by a CD8 hinge / transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta-activating domain. These two designs differ in the order of the CAR chains; in one design, the CD22 CAR comes first, followed by the 2A element and the tandem 2019 CAR, while in the other, it is the reverse. (Figure 10)

[0300] First, a set of four bicistronic DuoCAR designs that simultaneously target CD19, CD20, and CD22 antigens under the control of the EF1a promoter was constructed as described above (Set 1, Table 1 below).

[0301] [Table 1]

[0302] To promote optimal expression of the CD22-targeted CAR site in the DuoCAR configuration, one of the CD22-reactive scFv sequences, 16P8 or 16P17, was incorporated into the CD22-targeted CAR chain. CD22 scFv m971 was used as a comparison cell, and untransduced cells (UTD) were used as a CAR-negative control. Co-expression of the CD20-CD19-targeted CAR chain and the CD22-targeted CAR chain was observed as described above in 2A ribosomes. The expression was promoted by the Kip sequence. Individually encoded CAR chains were included as expression controls. Human primary T cells from healthy donors were transduced with lentiviral vectors encoding each DuoCAR or a single CAR control. CAR expression was examined by flow cytometry after T cell culture proliferation was complete. CAR20 in the DuoCAR group + CAR22 + The proportion of double-positive cells (representing co-expression of tandem CD20-CD19 CAR chains and CD22-CAR chains within the same cell) (LTG2515, LTG2520, LTG2521) was relatively low, ranging from 28% (LTG2515, LTG2520) to 9% (LTG2521) (Figure 11). In contrast, the expression of individual CAR controls was significantly higher, reaching up to 72% for the CD22-targeted construct (LTG2200) and up to 38% for the tandem CD20-CD19-targeted CAR (LTG2228) (Figure 11). Next, the functionality of DuoCAR was tested using a cytokine release assay. Control DuoCAR effector cells and Raji target cells were combined at an effector-target ratio (E:T) of 10 and left overnight. At the end of the incubation period, the cell culture supernatant was collected and assayed for the secretion of T cell cytokines IFN-gamma, TNF-alpha, and IL-2 (Figure 12). Effectors incubated under similar conditions except for the absence of tumor target cells were used as a further control for spontaneous cytokine release. When Raji tumor cells and CAR effectors were incubated together, IFN-gamma, IL-2, and TNF-alpha were strongly upregulated for all constructs. Notably, no spontaneous cytokine production occurred for any of the CARs. However, the degree of cytokine secretion tended to be lower for all Duo CAR constructs than for the positive controls CAR22 LTG2200 and tandem 2019 CAR LTG2228, which is thought to be due to the relatively low expression of DuoCAR, as shown in Figure 11.

[0303] The lower DuoCAR expression and cytokine response compared to the single CAR control (Figures 11 and 12) suggest that the large payload size unfavorably affects DuoCAR expression efficiency in this configuration. To improve DuoCAR transduction efficiency, the selected DuoCAR sequence was re-codon optimized as needed, and the expression cassette was re-cloned into a new expression backbone under the control of an MSCV internal promoter to improve bicistronic expression (Set 2, Table 1).

[0304] Lentiviral vectors were constructed for each of the new Duo CAR constructs and transduced into CAR T cells for proliferation (as described in Materials and Methods). DuoCAR expression was examined by flow cytometry. The percentage of CD19+CD22+ T cells represents cells co-expressing both strands of DuoCAR (Figure 13). High transduction efficiencies were achieved for Duo CAR constructs D0044 (MSCV_20-19-28z-2A-16p8-BBz) and D0047 (MSCV_16p8-BBz-2A-20-19-28z) (both anti-CD22 scFv). 16P8 (Figure 13, containing 51% and 45%, respectively). Unexpectedly, DuoCAR D0043, which contained the comparison m971 CD22 scFv in the farther orientation, expressed well (MSCV_20-19-28z-2A-m971-BBz, 46% positive), but did not express in the opposite orientation (D0046, MSCV_m971-BBz-2A-20-19-28z). Therefore, the selection of scFv sequences included in the DuoCAR design, sequence codon optimization, and expression backbone selection are all important for optimal DuoCAR expression.

[0305] The cytotoxic function of T cells transduced with DuoCAR set 2 was assessed using an overnight death assay against panel tumor lines, specifically by measuring the expression of tumor antigens CD19, CD20, and CD22. The cells were altered and assayed. All strains were stably transduced to express firefly luciferase and then subjected to cell death assays (as described in Materials and Methods). First, CD19+CD20+CD22+ DuoCARs were combined with non-Hodgkin lymphoma Raji, or acute lymphoblastic leukemia Reh cells, or CD19-CD20-CD22-human embryonic kidney 293 T cell lines (Figure 15). These included DuoCARs D0044 and D0047 that bicistronically encode CAR 20-19-28z and CD22 CAR 16p8-BBz CARs, as well as a single CAR 22 control LTG2200, a tandem CAR control 20-19 LTG1497, and an untransduced T cell control (UTD) (Figure 14). In the legend of the figures, constructs D0043, D0044, D0046, and D0047 are abbreviated as D43, D44, D46, and D47, respectively (Figure 14). Effector and target cells were incubated overnight in ratios of 2.5, 5, or 10 (3 cells each), then harvested from plates and luciferase activity of viable tumor cells was examined by luminometry using SteadyGlo reagent. Overall, CAR cell lysis function correlated with DuoCAR expression (Figure 13). DuoCAR D0047 and D0044, as well as the positive control DuoCAR D0043, very well lysed the CD19, CD20, and CD22 triple-positive tumor lines Raji and Reh, while the suboptimally expressed construct D0046 had relatively poor lysis function (Figure 14). None of the CAR constructs lysed the CD19-CD20-CD22 triple-negative strains, highlighting that CAR-mediated lysis is specific to congener antigens.

[0306] To further illustrate the specificity of the Duo CAR construct, recombinant K562 strains expressing either the antigen CD19, CD20, or CD22 were generated (referred to as K19, K20, and K22, respectively) (Figure 15). When incubated with single-positive tumor strains and assayed, DuoCAR D44 and D47 (containing CAR chains targeting CD19, CD20, and CD22) potently lysed each target strain in an effector target ratio-dependent manner, and their function was similar to that of the comparison partner DuoCAR D0043 (the construct names in the legend of the figures are abbreviated from D0043, D0044, D0046, and D0047 to D43, D44, D46, and D47, respectively, Figure 15). Control T cells expressing tandem2019 CAR(1497) lysed tumor lines K19 and K20, but their lytic effect against K22 was very weak and only in the background (less than 10% lysis at the best E:T ratio of 10). The single CD22 control CAR strongly lysed K22 tumor cells, but had no function against K20 cells and showed only background-level lysis against K19 cells (10% lysis at the best E:T ratio of 10:1). Therefore, this experimental system allows for highly accurate testing of CAR reactivity to each tumor antigen. In short, the functionality of each tumor-targeting domain of both DuoCAR D0044 and D0047 was demonstrated in this single-antigen expression test system (Figure 15).

[0307] To characterize the cytokine release response of the Duo CAR construct, DuoCAR T cell preparations D0044 and D0047 (D44 and D47 in the legend of the figures, respectively), which are CD19+, CD20+, and CD22+, were co-colonized with Raji tumor cells at an E:T ratio of 10 and left overnight. The culture supernatant was analyzed by ELISA to examine T cell cytokines IFNg, TNFα, and IL-2 (Figure 16). Single CAR22 construct LTG2200 and tandem 2019 CAR construct LTG2273 were included for comparison, and untransduced T cells (UTDs) were used as a negative control. In parallel, CAR T cells from each group were incubated under similar conditions except in the absence of tumor cells, and spontaneous cytokine release was examined (Figure 16). Spontaneous cytokine release did not occur in any of the constructs, but DuoCAR D Both D44 and D47 strongly induced IL-2, IFNg, and TNFα after incubation with the Raji target, highlighting the efficacy of these Duo CAR constructs. Notably, despite the simultaneous co-expression of the two chains within the same cell, no evidence of tonic signaling was detected, which was confirmed by the complete absence of spontaneous cytokine release (Figure 16).

[0308] We were able to develop a bicistronic DuoCAR consisting of two CAR chains with distinct and complementary co-stimulatory domains targeting three different tumor antigens CD19, CD20, and CD22. However, there was a question as to whether a similar construct could be created using other approaches. Bicistronic expression of distinct CAR chains within the same ORF requires multiple optimization and purification steps, and it is thought that each new sequence set must be unique. In contrast, combining two CAR sequences during lentiviral vector preparation or CAR T transduction could provide a more universal and rapid approach, allowing the creation of a CAR combination expressible in the same cell or the same T cell population by transduction of T cells using a single lentiviral preparation. In this example, similar to the DuoCAR approach, the first CAR chain is CD22 The scFv consists of an in-frame CD8 hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta-activating domain. The second CAR chain consists of a targeting domain based on a tandem CD20 CD19 scFv, followed by a CD8 hinge / transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta-activating domain (Figure 17). In the simultaneous transfection approach, during the vector preparation process, two transfer plasmids encoding one CAR chain each are mixed and bound to a helper plasmid according to a standard protocol (see Materials and Methods). Thus, the resulting lentiviral preparation encodes a mixture of the two CAR chains described above. Using this approach, a set of lentiviral preparations encoding two CAR chains simultaneously was prepared (Table 2 below).

[0309] [Table 2]

[0310] Transfer plasmids for CAR22 (D1, D2, and D3, respectively) utilizing the MSCV promoter-controlled scFv 16P17, 16P8, and 16P13 CAR22-4-1BB-CD3 zeta were constructed, along with the MSCV-controlled tandem CAR 2019-28-CD3 zeta (LTG2273). In parallel, lentiviral vectors encoding only each CAR strand were prepared. High titers were uniformly achieved for all DuoCAR co-transfection preparations (10 10 (TU / ml, not specified), this was intended to emphasize the efficiency of the approach.

[0311] To optimize DuoCAR function, a series of CAR22 constructs consisting of scFvs 16P17, 16P8, and 16P13 controlled by the MSCV promoter were designed (constructs D1, D2, and D3, respectively), and a tandem CAR 2019 (LTG2273), also controlled by the MSCV promoter, was used as a combination for DuoCAR co-transfection (Table 2 and Figure 18). Lentiviral vectors were prepared by co-transfection of LTG2273 with one of the CD22 CAR plasmids, yielding high infectivity titers (not described). Each LV was transduced into healthy donor T cells using an infection multiplicity (MOI) of 20, and CAR expression was examined by flow cytometry (Figure 18). All control groups transfected with LV encoding a single CAR control showed high CAR expression (D1: 45%, D2: 82%, D3: 82%, 2273: 87% (not specified)). Surprisingly and unexpectedly, in combination co-transfection, the D2+73 and D3+73 groups showed high efficiency, almost the same as the case of co-expression of two CAR chains (51%), but the D1+73 combination did not co-express (2.8% CAR+) (Figure 18). To investigate whether these DuoCARs have lytic function, CAR T cells from each group were tested against a tumor line panel (Figure 19; the "D" in the names of the D1+2273, D2+2273, and D3+2273 groups has been omitted for simplification). All DuoCAR preparations efficiently lysated the triple-positive tumor strains Raji and Reh, but not the triple-negative strain 293T, demonstrating the specificity of DuoCAR (Figure 19A). In addition, all DuoCARs showed stronger lysis than the background against single-antigen tumor strains K19, K20, and K22, but single control CARs with incompatible targeting domains did not show specific lysis (see D1-D3 for K19, D1-D3 for K20, and 2273 for K22) (Figure 19B).Next, we assayed the ability of DuoCAR to induce cytokines when incubated with specific tumor targets (Figure 20; the "D" in the names of the D1+2273, D2+2273, and D3+2273 groups has been omitted for simplicity). DuoCAR T cells, single CAR controls, and untransduced T cells (UTDs) were incubated overnight with triple CD19+CD20+CD22+ Raji tumor cells. In parallel, CAR T cells were incubated in the absence of tumor under similar conditions to exclude spontaneous cytokine release. At the end of the incubation period, the culture supernatant was assayed by ELISA for cytokines IFNg, TNFα, and IL-2 (Figure 20). All CAR groups showed high IFNg levels when incubated with Raji. On the other hand, several single CD22 CAR controls showed moderate spontaneous IFNg release (D2, D3), while none of the DuoCARs spontaneously produced IFNg, suggesting that the safety margin for DuoCARs may be even wider. Furthermore, incubation with Raji greatly induced IL-2 and TNFα expression in all CAR groups except CAR 2272 (Figure 20).

[0312] In short, this specification describes the creation of functional and highly specific DuoCARs by co-transfecting individual CAR chains during LV preparation and applying the resulting LV preparation to T cell transduction. Furthermore, by using recombinant cell lines expressing only one target antigen (K19, K20, K22), the inventors demonstrated that each CAR targeting domain possesses functionality and can induce DuoCAR function against target-expressing tumor cells. Surprisingly and unexpectedly, only two or three combinations were able to exhibit both strong CAR expression and highly potent cytotoxic function, thus demonstrating that DuoCAR design is not a trivial matter.

[0313] Sequence List Sequence ID 1 is the nucleotide sequence of the CD20-reactive scFv-binding domain (LTG1495): GAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAA CCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCT CCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCGTCACCCAAGCCT TGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTCAGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGACGCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACTAAGCTGGAGATCAAA Sequence ID 2 is the amino acid sequence of the CD20-reactive scFv binding domain (LTG1495): EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVS SGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIK Nucleotide sequence of sequence number 3 CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta): Amino acid sequence of sequence number 4 CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSN YYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYY CQQWSFNPPTFGGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 5 is the nucleotide sequence of the leader / signal peptide sequence: ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG Sequence ID 6 is the amino acid sequence of the leader / signal peptide sequence: MLLLVTSLLLCELPHPAFLLIP Sequence ID 7 is the nucleotide sequence of the CD22-reactive scFv-binding domain (LTG2200): CAGGTACAGCTCCAGCAGAGTGGCCCAGGGCTCGTGAAGCCAAGCCAGACGCTGTCCCTGACTTGTGCAATTTCAGGGGATTCAGTTTCATCAAATAGCGCGGCGTGGAATTGGATTCGACAATCTCCTTCCCGAGGGTTGGAATGGCTTGGACGAACATATTACAGATCCAAATGGTATAACGACTATGCGGTATCAGTAAAGTCAAGAATAACCATTAACCCCGACACAAGCAAGAACCAATTCTCTTTGCAGCTTAACTCTGTCACGCCAGAAGACACGGCAGTCTATTATTGCGCTCGCGAGGTAACGGGTGACCTGGAAGACGCTTTTGACATTTGGGGGCAGGGTACGATGGTGACAGTCAGTTCAGGGGGCGGTGGGAGTGGGGGAGGGGGTAGCGGGGGGGGAGGGTCAGACATTCAGATGACCCAGTCCCCTTCATCCTTGTCTGCCTCCGTCGGTGACAGGGTGACAATAACATGCAGAGCAAGCCAAACAATCTGGAGCTATCTCAACTGGTACCAGCAGCGACCAGGAAAAGCGCCAAACCTGCTGATTTACGCTGCTTCCTCCCTCCAATCAGGCGTGCCTAGTAGATTTAGCGGTAGGGGCTCCGGCACCGATTTTACGCTCACTATAAGCTCTCTTCAAGCAGAAGATTTTGCGACTTATTACTGCCAGCAGTCCTATAGTATACCTCAGACTTTCGGACAGGGTACCAAGTTGGAGATTAAGGCGGCCGCA SEQ ID NO: 8 is the amino acid sequence of the CD22-reactive scFv binding domain (LTG2200): QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAA Nucleotide sequence of sequence number 9 CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 zeta): Amino acid sequence of Sequence ID No. 10 CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 Zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR EVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATY YCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 11 is the nucleotide sequence of the DNA CD8 transmembrane domain: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC Sequence ID 12 is the amino acid sequence of the CD8 transmembrane domain: IWAPLAGTCGVLLLSLVITLYC Sequence ID 13 is the nucleotide sequence of the DNA CD8 hinge domain: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCCAGTGCACACGAGGGGGCTGGACTTTGCCTGCGATATCTAC Sequence ID 14 is the amino acid sequence of the CD8 hinge domain: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY Sequence ID 15 is the amino acid sequence of amino acid numbers 137-206 in the hinge / transmembrane region of CD8 alpha (NCBI RefSeq: NP.sub.--001759.3): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC Sequence ID 16 is the amino acid sequence of the human IgG CL sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Sequence ID 17 is the nucleotide sequence of the DNA signaling domain 4-1BB: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG Sequence ID 18 is the amino acid sequence of the signaling domain 4-1BB: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Sequence ID 19 is the nucleotide sequence of the DNA signaling domain CD3-zeta: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC Sequence ID 20 is the amino acid sequence of CD3 zeta: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 21 is the nucleotide sequence of CAR LTG1562 (LP-CD19 binder-CD8 linker-CD4tm-4-1BB-CD3-zeta): AAGGCGAACGCCGCCGCGGCAAAGGCCATGATGGCCTGTATCAGGGCCTGAGCACCGCGACCAAAGATACCTATGATGCG CTGCATATGCAGGCGCTGCCGCCGCGC Sequence ID 22 is the amino acid sequence of CAR LTG1562 (LP-CD19 binder-CD8 bond-CD4™-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSAAAPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFVQPMALIVLGGVAGLLLFIGLGIFFCVRCRPRRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 23 is the nucleotide sequence of the CD20_19 reactive scFv binding domain (LTG1497 bispecific binder): Sequence ID 24 is the amino acid sequence of the CD20_19 reactive scFv binding domain (LTG1497 bispecific binder): EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKGGGGSGGGGSGGGS GGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAA Sequence ID 25 is the nucleotide sequence of CAR LTG1497 (LP-LTG1497-CD8 TM-41BB-CD3 zeta, or LP-CD20 VH-(GGGGS)3-CD20 VL-(GGGGS)5-CD19VL-Whitlow linker-CD19 VH-CD8 hinge+TM-41BB-CD3 zeta): ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCTGCCTTCCTGCTGATTCCCGAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTC Sequence ID 26 is the amino acid sequence of CAR LTG1497 (LP-LTG1497-CD8 TM-41BB-CD3 zeta, or LP-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH-CD8 hinge+TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 27 is the nucleotide sequence of scFV relative to CD19: GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 28 is the amino acid sequence of the scFV against CD19: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS Sequence ID 29 is the nucleotide sequence of CAR LTG 1494 (LP-CD19 binder-CD8 binding-CD8™-41BB-CD3 zeta): Sequence ID 30 is the amino acid sequence of CAR LTG1494 (LP-CD19 binder-CD8 binding-CD8™-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPDTDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 31 is the nucleotide sequence of CAR LTG1538 (LP-CD19 binder-CD8 binding-CD8™-signal (LTI redesigned CD19 CAR)): CAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGAC GCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Sequence ID 32 is the amino acid sequence of CAR LTG1538 (LP-CD19 binder-CD8 binding-CD8™-signal (LTI redesigned CD19 CAR)): MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 33 is the nucleotide sequence of the CD19_20 reactive scFv binding domain (LTG1496): Sequence ID 34 is the amino acid sequence of the CD19_20 reactive scFv binding domain (LTG1496): DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKL QESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGGSGGGGSGGG GSGGGGSGGGGSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKAAA Sequence ID 35 is the nucleotide sequence of CAR LTG1496 (LP-LTG1496-CD8 TM-41BB-CD3 zeta, or LP-CD19 VL-Whitlow linker-CD19 VH(GGGGS)5CD20 VH(GGGGS)3-CD20 VL CD8 hinge+TM-41BB-CD3 zeta): Sequence ID 36 CAR LTG1496 (LP-LTG1496-CD8 TM-41BB-CD3 Zeta) Alternatively, the amino acid sequence of LP-CD19 VL-Whitlow linker-CD19 VH-(GGGGS)5-CD20 VH(GGGGS)3-CD20 VL-CD8 hinge + TM-41BB-CD3 zeta: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS EDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 37 is the nucleotide sequence of the mesothelin-reactive scFv-binding domain (LTG1904): GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGA ATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTT TAACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGCGGATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGAC AGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT Sequence ID 38 is the amino acid sequence of the mesothelin-reactive scFv-binding domain (LTG1904): EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGG GGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG Nucleotide sequence of sequence number 39 CAR LTG1904 (LP-LTG1904-CD8 TM-41BB-CD3 zeta): Amino acid sequence of sequence number 40 CAR LTG1904 (LP-LTG1904-CD8 TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLS SVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS RDSSGNHLVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 41 is the nucleotide sequence of the CD33-reactive single-strand binding domain VH-4 (LTG1906): GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGCCAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTG AGAAATACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAAGAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA Sequence ID 42 is a CD33-reactive single-strand binding domain VH-4 (LTG1906). It is a amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSS Sequence ID 43 is the nucleotide sequence of CAR LTG1906 (LP-VH4-CD8 TM-41BB-CD3 zeta): Sequence ID 44 is the amino acid sequence of CAR LTG1906 (LP-VH4-CD8 TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 45 is the nucleotide sequence of the TSLPR-reactive scFv-binding domain (LTG1789): ATGGCACTGCCCGTGACCGCCCTGCTTCTGCCGCTTGCACTTCTGCTGCACGCCGCTAGGCCCCAAGTCACCCTCAAAGAGTCAGGGCCAGGAATCCTCAAGCCCTCACAGACTCTGTCTCTTACTTGCTCATTCAGCGGATTCAGCCTTTCCACCTCTGGTATGGGCGTGGGGTGGATTAGGCAACCTAGCGGAAAGGGGCTTGAATGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCTCACTGAAGTCCCAGCTCACTATTTCCAAAGATACTTCCCGGAATCAGGTGTTCCTCAAGATTACCTCTGTCGACACCGCTGATACCGCCACTTACTATTGTTCACGCAGACCGAGAGGTACCATGGACGCAATGGACTACTGGGGACAGGGCACCAGCGTGACCGTGTCATCTGGCGGTGGAGGGTCAGGAGGTGGAGGTAGCGGAGGCGGTGGGTCCGACATTGTCATGACCCAGGCCGCCAGCAGCCTGAGCGCTTCACTGGGCGACAGGGTGACCATCAGCTGTCGCGCATCACAAGATATCTCTAAGTATCTTAATTGGTACCAGCAAAAGCCGGATGGAACCGTGAAGCTGCTGATCTACTACACCTCACGGCTGCATTCTGGAGTGCCTAGCCGCTTTAGCGGATCTGGGTCCGGTACTGACTACAGCCTCACCATTAGAAACCTTGAACAGGAGGACATCGCAACTTATTTCTGCCAACAGGTCTATACTCTGCCGTGGACCTTCGGCGGAGGTACCAAACTGGAGATTAAGTCCGG SEQ ID NO: 46 is the amino acid sequence of the TSLPR-reactive scFv binding domain (LTG1789): MALPVTALLLPLALLLHAARPQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCSRRPRGTMDAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAASSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQQVYTLPWTFGGGTKLEIKS Sequence ID 47 is the nucleotide sequence of CAR LTG1789 (LP-3G11-CD8 TM-41BB-CD3 zeta): ATGGCACTGCCGTGACCGCCCTGCTTCTGCCGCTTGCACTTCTGCTGCACGCCGCTAGGCCCCAAGTCACCCTCAAAGAGTCAGGGCCAGGAATCCTCAAGCCCTCACAGACTCTGTCTCTTACTTGCTCATTCACGGATTCAGCCTTTCCACCTCTG GTATGGGCGTGGGGGTGGATTAGGCAACCTAGCGGAAAGGGGCTTGAATGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCTCACTGAAGTCCCAGCTCACTATTTCCAAAGATACTTCCCGGAATCAGGTGTTCCTCAAGATTACCTC TGTCGACACCGCTGATACCGCCACTTACTATTGTTCACGCAGACCGAGAGGTACCATGGACGCAATGGACTACTGGGGACAGGGCACCAGCGTGACCGTGTCATCTGGCGGTGGAGGGTCAGGAGGTGGAGGTAGCGGAGGCGGTGGGTCCGACATTGTCATGACCCAGGCCGCCAGCAGCCTGAGCGCTTCACTGGGCGACAGGGTGACCATCAGCTGTCGCGCATCACAAGATATCTCTAAGTATCTTAATTGGTACCAGCAAAAGCCGGATGGAACCGTGAAGCTGCTGATCTACTACACCTCACGGCTGCATTCTGGAGTGCCTAGCCGCTTTAGCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 48 is the amino acid sequence of CAR LTG1789 (LP-3G11-CD8 TM-41BB-CD3 zeta): MALPVTALLLPLALLLHAARPQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCSRRP RGTMDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAASSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFC QQVYTLPWTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 49 is the nucleotide sequence of CAR LTG1563 (LP-CD19-TNFRSF19TM-41BB-CD3 zeta): Sequence ID 50 is the amino acid sequence of CAR LTG1563 (LP-CD19-TNFRSF19TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSAAAPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQPRRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 51 is the nucleotide acid sequence of CAR LTG2228 (LP-CD20_CD19-CD8TM-CD28-CD3 zeta): Sequence ID 52 is the amino acid sequence of CAR LTG2228 (LP-CD20_CD19-CD8™-CD28-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 53 D0043 nucleotide sequence ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCTGCCTTCCTGCTG ATTCCCGAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTG AAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAA CAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACT TCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACC GCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGG TCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTG ACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGG ATCCGAC ATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATG ACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCG TCACCCAAGCCTTGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTC AGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGAC GCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACT AAGCTGGAGATCAAAGGAGGCGGCGGCAGCGGCGGGGGAGGGTCCGGAGGGGGTGGTTCT GGTGGAGGAGGATCGGGAGGCGGTGGCAGCGACATTCAGATGACTCAGACCACCTCCTCC CTGTCCGCCTCCCTGGGCGACCGCGTGACCATCTCATGCCGCGCCAGCCAGGACATCTCG AAGTACCTCAACTGGTACCAGCAGAAGCCCGACGGAACCGTGAAGCTCCTGATCTACCAC ACCTCCCGGCTGCACAGCGGAGTGCCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGAC TACTCCCTTACTATTTCCAACCTGGAGCAGGAGGATATTGCCACCTACTTCTGCCAACAA GGAAACACCCTGCCGTACACTTTTGGCGGGGGAACCAAGCTGGAAATCACTGGCAGCACA TCCGGTTCCGGGAAGCCCGGCTCCGGAGAGGGCAGCACCAAGGGGGAAGTCAAGCTGCAG GAATCAGGACCTGGCCTGGTGGCCCCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCC GGAGTGTCGCTCCCGGATTACGGAGTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTG GAATGGCTCGGAGTCATCTGGGGTTCCGAAACCACCTATTACAACTCGGCACTGAAATCC AGGCTCACCATTATCAAGGATAACTCCAAGTCACAAGTGTTCCTGAAGATGAATAGCCTG CAGACTGACGACACGGCGATCTACTATTGCGCCAAGCACTACTACTACGGCGGATCCTAC GCTATGGACTACTGGGGCCAGGGGACCAGCGTGACCGTGTCATCCGCGGCCGCGACTACC ACTCCTGCACCACGGCCACCTACCCCAGCCCCCACCATTGCAAGCCAGCCACTTTCACTG CGCCCCGAAGCGTGTAGACCAGCTGCTGGAGGAGCCGTGCATACCCGAGGGCTGGACTTC GCCTGTGACATCTACATCTGGGCCCCATTGGCTGGAACTTGCGGCGTGCTGCTCTTGTCT CTGGTCATTACCCTGTACTGCCGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATG AACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCT CGGGATTTCGCCGCATACCGGTCCAGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCC TACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATAT GACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAG AACCCTCAAGAGGGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCC GAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGGG CGCGCGAAACGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTG GAAGAAACCCGGCCCGGAGCAAAGGAATATTATGCTTATTAGTGACTTCCCTT TTGCTGTGCGAGTTGCCACACCCCGCCTTCTGCTTATTCCCAGGTACAGCTCCAGC AGTGGCCAGGGCTCGTGAAGCCAAGCCAAGCGCGTCTCCTACTTGTGCAATTTCAGGG GATTCAGTTTCATCAAATAGCGCGGCGTGGAATTGGATTCGACAATCTCCTTCCCGAGGGG TTGGAATGGCTTGGACGAACATATTACAGATGGTATAACGACTATGCGGTATCA GTAAAGTCAAGAATAACCATTAACCCCGACACACAAGCAAGAACCAATTCTCTTGCAGCTT AACTCTGTCAGCCGCAGAAGACAGGCCAGTCTATTTGCGCTCGGAGGTAACGGGTGAC CTGGAAGAGCGCTTTTTGACATTTGGGGCAGGGGTACA GGTGGGAGTGGGGGAGGGTAGGCGGGGGGGAGGGTCAGACATTCAGATGACCCAGTCCC CCTTCATCCTTGTCTGCCTCCGTCGGTGACAGGGTGACAACATGCAGAGCAAGCCAA ACAATCTGGAGCTATCTCAACTGGTACCAGCAGCGACCAGGAAAAGCGCCAAACCTGCTG ATTTACGCTGCTTCCTCCCTCCAATCAGGCGTGCCTAGTAGATTTAGCGGTAGGGCTCC GGCACCGATTTTACGCTCACTATAAGCTCTTCCAAGCAGAAGATTTTGCGACTTATTAC TGCCAGCAGTCCTATAGTATACCTCAGACTTTCGGACAGGGTACCAAGTTGGAGATTAAGGCTAGCGCAACCACTACGCCTGCTCCGGCGGCCTCCAACGCCGCGCCCCACGATAGCTAGT CAGCCGTTGTCTCTCCGACCAGAGCGTGATACCGGCCGCTGGCGGAGCCGTACATACT CGCGGACTCGACTTCGCTTGCGACATCTACATTTGGGCACCCTTGGCTGGGACCTGTGGG GTGCTGTTGCGTCCTTGGTTATTACGTTGTACTGCAAGAGGGGCCGGAAGAAGCTGCTT TACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGCAGACTAGGAAGAGGACGGATGC TCGTGCAGATTCCCTGAGGAGGAAGGGGGGATGCGAACTGAGAGTCAAATTTTCCAGG TCCGCAGATGCCCCCGCGTACCAGCAAGGCCAGAACCACTTTACAACGAACTGAACCTG GGTCGCCGGGAGGAATATGTGCTGGATAAACGAAGGGGGAGGACCCTGAGATGGGA GGGAAACCTCGCAGGAAAACCCGCAGGGATTGTACAACGAGTTGCAGAAGGATAAG ATGGCTGAGGCTTACTCTGAAATAGGGAAGGGAGAGACGGGAGAAAAGGCCAT GATGGCCTTTACCAGGGCTTGAGCACAGCAACAAAGGATACTTACGACGCTCTTCACATG CAAGCTCTGCCACCACGG குற்றுக்க்குக்குக்கு54 D0043 அயையுக்க்குக்குகு MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGST SGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPRRAKRGSGATNFSLLKQAGDVEENPGPRAKRNIMLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSV SSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGS DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKASAT TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 55 D0044 nucleotide sequence CCAGCAGAAGCCAGGCTTGGCTCCTCAACTGCTGATCTTCGGCGCCAGCACTCTTCAGGGGGAAGTGCCATCACGCTTCTCCGGATCCGGTTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCTGAGGACTTCGCCACTTACTACTGCCAACAGGCCAAGTACTTCCCCTATACCTTCGGAAGAGGCACTAAGCTGGAAATCAAGGCTAGCGCAACCACTACGCCTGCTCCGCGGCCTCCAACGCCCGCGCCCACGATAGCTAGTCAGCCGTTGTCTCTCCGACCAGAGGCGTGTAGACCGGCCGCTGGCGGAGCCGTACATACTCGCGGACTCGACTTCGCTTGCGACATCTACATTTGGGCACCCTTGGCTGGGACCTGTGGGGTGCTGTTGCTGTCCTTGGTTATTACGTTGTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGAGAGTCAAATTTTCCAGGTCCGCAGATGCCCCCGCGTACCAGCAAGGCCAGAACCAACTTTACAACGAACTGAACCTGGGTCGCCGGGAGGAATATGATGTGCTGGATAAACGAAGGGGGAGGGACCCTGAGATGGGAGGGAAACCTCGCAGGAAAAACCCGCAGGAAGGTTTGTACAACGAGTTGCAGAAGGATAAGATGGCTGAGGCTTACTCTGAAATAGGGATGAAGGGAGAGAGACGGAGAGGAAAAGGCCATGATGGCCTTTACCAGGGCTTGAGCACAGCAACAAAGGATACTTACGACGCTCTTCACATGCAAGCTCTGCCACCACGG SEQ ID NO: 56 D0044 Amino Acid Sequence Sequence ID 59 D0046 nucleotide sequence Sequence ID 60 D0046 Amino Acid Sequence Sequence ID 61 D0047 nucleotide sequence ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCA Sequence ID 62 D0047 Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSK Sequence ID 65 D0001 nucleotide sequence Sequence ID 66 D0001 Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 67 D0002 nucleotide sequence ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTGACTTGCGCAATTAGCGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAG GGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTATACCGACTACGCCGTGTCCGTGAAGAATCGGATCACCATTAACCCCGACACCTCGAAGAACCAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCACAGAAGTGGAACCGCAGGACGCCTTCGACATTT Sequence ID 68 D0002 Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYTDYAVSVKNRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGRGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 69 D0003 nucleotide sequence Sequence ID 70 D0003 Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGNSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGGGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 73 LTG2273 nucleotide sequence Sequence ID 74, 2273 amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 75 LTG2200 nucleotide sequence ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCCCAGGTACAGCTCCAGCAGAGTGGCCCAGGGCTCGTGAAGCCAAGCCAGACGCTGTCCCTGACTTGTGCAATTTCAGGGGATTCAGTTTCATCAAATAGCGCGGCGTGGAATTGGATTCGACAATCTCCTTCCCGAGGGTTGGAATGGCTTGGACGAACATATTACAGATCCAAATGGTATAACGACTATGCGGTATCAGTAAAGTCAAGAATAACCATTAACCCCGACACAAGCAAGAACCAATTCTCTTTGCAGCTTAACTCTGTCACGCCAGAAGACACGGCAGTCTATTATTGCGCTCGCGAGGTAACGGGTGACCTGGAAGACGCTTTTGACATTTGGGGGCAGGGTACGATGGTGACAGTCAGTTCAGGGGGCGGTGGGAGTGGGGGAGGGGGTAGCGGGGGGGGAGGGTCAGACATTCAGATGACCCAGTCCCCTTCATCCTTGTCTGCCTCCGTCGGTGACAGGGTGACAATAACATGCAGAGCAAGCCAAACAATCTGGAGCTATCTCAACTGGTACCAGCAGCGACCAGGAAAAGCGCCAAACCTGCTGATTTACGCTGCTTCCT CCCTCCAATCAGGCGTGCCTAGTAGATTTAGCGGTAGGGGCTCCGGCACCGATTTTACGCTCACTATAAGCTCTCTTCAAGCAGAAGATTTTGCGACTTATTACTGCCAGCAGTCCTATAGTATACCTCAGACTTTCGGACAGGGTACCAAGTTGGAGATTAAGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 76 LTG2200 Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR EVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATY YCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 77 GMCSF Reader Peptide Nucleotide Sequence ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCC Sequence ID 78 GMCSF Leader Peptide Amino Acid Sequence MLLLVTSLLLCELPHPAFLLIP Sequence ID 79 CD8a Reader Peptide Nucleotide Sequence ATGGCCCTGCCCGTCACTGCGCTGCTTCTTCCACTTGCGCTTCTGCTGCACGCAGCGCGCCCG Sequence ID 80 CD8a Leader Peptide Amino Acid Sequence MALPVTALLLPLALLLHAARP Sequence ID 81: CD8 hinge / transmembrane domain nucleotide sequence GCGGCCGCTACCACAACCCCTGCGCCCCGGCCTCCTACCCCCGCACCCACGATTGCTTCTC AACCTCTTTCACTCCGACCTGAGGCTTGTAGACCTGCAGCCGGGGGTGCCGTCCACACAC GGGGACTCGACTTCGCTTGTGATATATATATTTGGGCGCCCCTGGCCGGCACTTGTGGAG TTCTTTTGCTCTCTCTTGTTATCACATTGTACTGC Sequence ID 82 CD8 hinge / transmembrane domain amino acid sequence AAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYC Sequence ID 83 4-1BB / CD137 costimulatory domain nucleotide sequence AAGCGAGGTAGGAAGAAATTGCTTTACATTTTTAAGCAGCCGTTCATGCGACCAGTACAG ACTACTCAAGAAGAAGATGGGTGCTCTTGTCGGTTCCCGGAAGAAGAAGAGGGTGGTTGC GAGTTG SEQ ID NO: 84 4-1BB / CD137 Co-stimulatory Domain Amino Acid Sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Sequence ID 85 CD28 costimulatory domain nucleotide sequence CGGTCGAAGCGCTCAAGACTGCTGCACTCAGACTACATGAACATGACTCCTCGGCGGCCG GGGCCGACTCGGAAGCACTACCAGCCTTACGCACCCCCGAGAGATTTCGCGGCCTACCGC TCC Sequence ID 86 CD28 Co-stimulatory Domain Amino Acid Sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Sequence ID 87 CD3 zetanucleotide sequence AGGGTGAAGTTCTCCCGCTCTGCCGACGCACCGGCATATCAGCAGGGACAAAACCAGCTC TACAACGAATTGAACCTGGGTCGGCGGGAAGAATATGACGTGCTCGATAAGCGGCGGGGT CGCGACCCAGAAATGGGAGGCAAACCGCGCAGGAAAAATCCACAGGAGGGACTTTATAAC GAACTTCAAAAGGATAAGATGGCAGAGGCATACAGCGAAATCGGGATGAAAGGCGAGAGA AGAAGGGGGAAAGGGCACGATGGTCTTTACCAGGGGCTTTCTACCGCGACGAAGGATACC TACGATGCTCTCCATATGCAAGCACTTCCTCCTAGA Sequence ID 88 CD3 zeta amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 89: Furin P2A furin nucleotide sequence CGGGCAAAGCGGGGCTCAGGGGCGACTAACTTTTCACTGTTGAAGCAGGCCGGGGATGTG GAGGAGAATCCTGGTCCTAGAGCTAAGCGAG Sequence ID 90: Furin P2A furin amino acid sequence RAKRGSGATNFSLLKQAGDVEENPGPRAKR Sequence ID 95 16P17 CD22 scFv VH nucleotide sequence CAGGTACAGCTTCAACAGAGTGGGCCGGGACTGGTGAAACACTCCCAAACACTTTCTCTG ACGTCGCTATATCAGGTGACTCTGTTTCATCTAATTCTGCTGCGTGGAACTGGATTCGA CAATCTCCCAGTCGCGGGTTGGAATGGCTGGGACGAACATATTATCGGTCTAAGTGGTAT AACGATTATGCTGTATCTGTTAAATCTCGAATTACGATTAATCCTGACACCTCCAAGAAC CAGTTCTCCCTCCAGTTGAACTCAGTCACACCGGAAGACACTGCGGTCTACTATTGCGCT CAAGAAGTCGAGCCACATGATGCATTCGACATCTGGGGCCAGGGAACGATGGTCACCGTC AGCAGT Sequence ID 96 16P17 CD22 scFv VH amino acid sequence QVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTV SS Sequence ID 97 16P17 CD22 scFv VL nucleotide sequence GACATACAAATGACGCAGAGTCCCTCAAGTGTGTACGCGAGTGTGGGGATAAGGTAACT ATTACGTGCAGAGCGTCACAGGATGTTAGTGGATGGCTTGCCTGGTATCAGCAGAAGCCA GGCCTTGCTCCACAGCTCCTTATCAGTGGTGCTTCTACACTTCAGGGCGAGGTTCCGAGT AGATTCTCTGGTTCTGGATCTGGTACTGACTTCACTCTTACAATTTCTTCTTTGCAACCA GAAGACTTTGCGACTTATTACTGCCAACAGGCCAAATACTTCCCTTATACATTTGGCCAA GGTACCAAGTTGGAGATAAAG Sequence ID 98 16P17 CD22 scFv VL amino acid sequence DIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence ID 99 16P8 CD22 scFv VH nucleotide sequence CAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTG ACTTGCGCAATTAGCGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGG CAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTAT ACCGACTACGCCGTGTCCGTGAAGAATCGGATCACCATTAACCCCGACACCTCGAAGAAC CAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCA CAAGAAGTGGAACCGCAGGACGCCTTCGACATTTGGGGACAGGGAACGATGGTCACAGTG TCGTCC Sequence ID 100 16P8 CD22 scFv VH amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY TDYAVSVKNRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTV SS Sequence ID 101 16P8 CD22 scFv VL nucleotide sequence GATATCCAGATGACCCAGAGCCCCTCCTCGGTGTCCGCATCCGTGGGCGATAAGGTCACC ATTACCTGTAGAGCGTCCCAGGACGTGTCCGGATGGCTGGCCTGGTACCAGCAGAAGCCA GGCTTGGCTCCTCAACTGCTGATCTTCGGCGCCAGCACTCTTCAGGGGGAAGTGCCATCA CGCTTCTCCGGATCCGGTTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCT GAGGACTTCGCCACTTACTACTGCCAACAGGCCAAGTACTTCCCCTATACCTTCGGAAGA GGCACTAAGCTGGAAATCAAG Sequence number 102, 16P8 CD22 scFv VL amino acid sequence DIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGRGTKLEIK Sequence number 103, 16P13 CD22 scFv VH nucleotide sequence CAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTG ACTTGCGCCATTAGCGGGAACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGG CAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTAT AACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCATTAACCCCGACACCTCGAAGAAC CAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCA CAAGAAGTGGAACCGCAGGACGCCTTCGACATTTGGGGACAGGGAACGATGGTCACAGTG TCGTCC Sequence ID 104 16P13 CD22 scFv VH amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGNSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTV SS Sequence ID 105 16P13 CD22 scFv VL nucleotide sequence GATATCCAGATGACCCAGAGCCCCTCCTCGGTGTCCGCATCCGTGGGCGATAAGGTCACC ATTACCTGTAGAGCGTCCCAGGACGTGTCCGGATGGCTGGCCTGGTACCAGCAGAAGCCA GGCTTGGCTCCTCAACTGCTGATCTTTGGCGCCAGCACTCTTCAGGGGGAGGTGCCATCA CGCTTCTCCGGAGGTGGTTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCT GAGGACTTCGCCACTTACTACTGCCAACAGGCCAAGTACTTCCCCTATACCTTCGGACAA GGCACTAAGCTGGAAATCAAG Sequence ID 106 16P13 CD22 scFv VL amino acid sequence DIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPS RFSGGGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence ID No. 107 Whitlow Linker Amino Acid Sequence GSTSGSGKPGSGEGSTKG Sequence ID 108: Movable interchain linker amino acid sequence GGGGSGGGG SGGGGSGGGG SGGGGS

Claims

1. A composition for immunotherapy of tumors expressing CD22, CD20, and CD19, The present invention comprises at least one polycistronic vector containing a promoter operably bound to a polycistronic nucleic acid sequence, The aforementioned polycistronic nucleic acid sequence encodes two or more functional CARs, each containing an extracellular antigen-binding domain, a transmembrane domain, and one or more non-identical intracellular signaling motifs, and each containing non-identical amino acid sequences independently selected from the group consisting of the amino acid sequences of SEQ ID NOs. 54, 56, and 62. An immunotherapy composition wherein the at least one polycistronic vector is used to genetically modify one or more lymphocyte populations.

2. A pharmaceutical composition for immunotherapy of tumors expressing CD22, CD20, and CD19, comprising a population of human lymphocytes in an antitumor-effective quantity, wherein each cell in the population of human lymphocytes comprises (a) at least one polycistronic vector, (b) each of the at least one polycistronic vectors encodes two or more functional CARs comprising non-identical amino acid sequences independently selected from the group consisting of the amino acid sequences of SEQ ID NOs. 54, 56, and 62, (c) each comprising an extracellular antigen-binding domain, a transmembrane domain, at least one linker domain, and one or more non-identical intracellular signaling motifs, and (d) the extracellular antigen-binding domain, the transmembrane domain, the at least one linker domain, and the one or more intracellular signaling motifs are covalently linked in each of the at least one polycistronic vectors, and the at least one polycistronic vector is used to genetically modify one or more lymphocyte populations.

3. The pharmaceutical composition according to claim 2, wherein the population of human lymphocytes is a population of T cells of a person having leukemia or lymphoma.

4. The pharmaceutical composition according to claim 3, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

5. The pharmaceutical composition according to claim 3, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

6. The pharmaceutical composition according to claim 2, wherein the population of human lymphocytes is T cells of a human having multiple myeloma.

7. The pharmaceutical composition according to claim 2, wherein the population of human lymphocytes is a population of human T cells having adult cancer selected from the group consisting of oral and pharyngeal cancer, gastrointestinal cancer, respiratory cancer, bone and joint cancer, soft tissue cancer, skin cancer, central nervous system cancer, breast cancer, reproductive organ cancer, urinary tract cancer, eye and orbital cancer, endocrine cancer, and brain cancer.

8. An immunotherapeutic composition for tumors expressing CD22, CD20, and CD19, comprising one or more isolated nucleic acid molecules encoding at least two vectors, wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, or 62, thereby resulting in the expression of two or more non-identical binding domains as a result of the combination of the at least two vectors.

9. The immunotherapeutic composition according to claim 8, wherein each vector encodes one or more functional CARs comprising the amino acid sequence of SEQ ID NO: 54, 56, or 62.

10. The immunotherapy composition according to claim 8, wherein each of the at least two vectors is an RNA vector or a DNA vector.

11. Use of nucleic acid molecules encoding two or more vectors for producing a pharmaceutical composition for treating mammals having a disease, disorder, or condition associated with high expression of tumor antigens CD22, CD20, or CD19, wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, or 62, and the combination of the two or more vectors is used to genetically modify one or more lymphocyte populations.

12. The use according to claim 11, wherein the genetically modified lymphocytes are autologous or allogeneic T cells, and the autologous or allogeneic lymphocytes are returned to the subject by direct injection.

13. The use according to claim 11, wherein the genetically modified lymphocytes are autologous T cells, and the autologous T cells are returned to the subject by direct injection to promote the in vivo proliferation and persistence of target-specific antitumor T cells, and as a result bring about target-specific stabilization, reduction, removal, remission, or elimination of cancer or cancer recurrence.

14. The use according to claim 11, wherein the one or more lymphocyte populations express activation or memory-related surface markers.

15. The use according to claim 11, wherein the one or more lymphocyte populations include T cells and dendritic cells obtained from a hematopoietic stem cell donor.

16. The use according to claim 11, wherein the disease is blood cancer.

17. The use according to claim 16, wherein the blood cancer is leukemia, lymphoma, or multiple myeloma.

18. The use according to claim 17, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

19. The use according to claim 17, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

20. The use according to claim 11, wherein the disease is an inflammatory disease or an autoimmune disease.

21. The use according to claim 20, wherein the inflammatory disease is graft-versus-host disease, cutaneous allergy, Alzheimer's disease, multiple sclerosis, Parkinson's disease, traumatic brain injury, or chronic traumatic encephalopathy.

22. The use according to claim 11, wherein the disease is chronic obstructive pulmonary disease.

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