Nucleic acid molecule, polypeptide, vector, cell, in vitro method, pharmaceutical composition, use, and method for treating cancer

Abstract

The present invention relates to nucleic acid molecules that encode a chimeric antigen receptor (CAR). The present invention also relates to a polypeptide of the chimeric antigen receptor (CAR). The present invention also relates to a vector comprising the nucleic acid molecules defined above. The present invention also relates to the cell that expresses the polypeptide defined above. The present invention also relates to an in vitro method for obtaining a cell. The present invention also relates to a pharmaceutical composition comprising the nucleic acid molecule, vector, polypeptides or cells as defined above and a pharmaceutically acceptable vehicle. The present invention also relates to use of the nucleic acid molecule, vector, polypeptide or composition, as defined above, for the treatment of cancer. The present invention also relates to use of the nucleic acid molecule, vector, polypeptide or composition, as defined above, for the production of a drug for the treatment of cancer. The present invention also relates to a method for treating cancer in a patient.

Description

NUCLEIC ACID MOLECULE, POLYPEPTIDE, VECTOR, CELL, IN VITRO METHOD, PHARMACEUTICAL COMPOSITION, USE, AND METHOD FOR TREATING CANCER FIELD OF THE INVENTION

[0001] The present invention is in the field of immunology. The present invention relates to nucleic acid molecules encoding a chimeric antigen receptor (CAR). The present invention also relates to a chimeric antigen receptor (CAR) polypeptide. The present invention also relates to a vector comprising the nucleic acid molecules defined above. The present invention also relates to the cell expressing the polypeptide defined above. The present invention also relates to an in vitro method for obtaining a cell. The present invention also relates to a pharmaceutical composition comprising the nucleic acid molecule, vector, polypeptides, or cells as defined above, and a pharmaceutically acceptable carrier. The present invention also relates to the use of the nucleic acid molecule, vector, polypeptide, or composition, as defined above, for the treatment of cancer.The present invention further relates to the use of the nucleic acid molecule, vector, polypeptide or composition, as defined above, for the production of a medicament for the treatment of cancer. The present invention further relates to a method for treating cancer in a patient BACKGROUND OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION.

[0002] Cancer is a global public health problem. In 2018, according to the World Health Organization, 18.1 million cases were diagnosed and 9.6 million people died from this disease worldwide. This is equivalent to one in six deaths worldwide (BRAY et al., 2018). In Brazil, INCA (National Cancer Institute) estimates 650,000 new cases for each year of the 2020-2022 triennium. The mortality rate was 16.6% in 2017, representing the second leading cause of death in the country. These data emphasize that investments in research to reduce cancer-related incidence and mortality are extremely important both to ensure the well-being of citizens and to reduce the significant economic impact of this disease (estimated at US$1.16 trillion in 2010).

[0003] Despite advances in cancer diagnosis and treatment, efforts to reduce the socioeconomic impact of this disease have been limited by the development of few alternative or complementary therapeutic strategies to conventional therapies, to which most neoplasms are resistant. However, studies in B-cell neoplasms have, over the last decade, supported the development of cellular immunotherapy using genetically modified cells. This therapeutic strategy is disruptive because it inaugurated a new treatment modality: the artificial genetic reprogramming of immunocompetent cells aimed at the direct destruction of tumor cells. B-cell neoplasms have been and continue to be one of the best models for the development and refinement of new advanced immunotherapy strategies. Therefore, they were used in the present invention.

[0004] B-cell neoplasms result from the oncogenic transformation of B-lineage cells at different stages of differentiation, which in turn implies different pathophysiologies. These neoplasms are divided into B-cell lymphomas and leukemias. Lymphomas originate from the oncological transformation of more mature B lymphocytes, whose uncontrolled proliferation leads to the generation of solid tumors primarily in the lymphoid organs (lymph nodes, spleen), but can also colonize other organs, including the bone marrow. Although there are exceptions, the hallmark of lymphomas is the formation of solid tumors without massive circulation of neoplastic cells. On the other hand, B-cell leukemias are composed of circulating neoplastic cells.

[0005] B-cell lymphomas account for 85% of all non-Hodgkin lymphomas (NHL), and 30% of these patients are diagnosed with the diffuse large B-cell lymphoma (DLBCL) subtype. DLBCL is an aggressive cancer that can spread rapidly throughout the body, requiring immediate treatment after diagnosis. The first line of treatment consists of conventional chemotherapy, radiotherapy, and possible use of monoclonal antibodies against antigens expressed on the surface of neoplastic cells (e.g., rituximab, anti-CD20). In some cases, hematopoietic stem cell transplantation (HSCT) may also be performed. However, half of lymphoma patients do not respond to or experience disease recurrence after conventional therapies.

[0006] Among B-cell leukemias, acute lymphoblastic leukemia (ALL) is derived from immature B cells and progresses rapidly. Chronic lymphocytic leukemia (CLL), in turn, has a more mature B-cell phenotype and progresses more slowly. The first-line treatment for these leukemias is conventional chemotherapy. However, in cases where patients relapse, immunotherapy using monoclonal antibodies against antigens expressed on the surface of neoplastic cells (e.g., rituximab, anti-CD20; alemtuzumab, anti-CD52) and allogeneic hematopoietic stem cell transplantation (HSCT) are used, the latter not being available to all patients due to poor donor compatibility. Furthermore, HSCT can be associated with complications such as infections, relapses of previous disease, and graft-versus-host disease (GVHD).

[0007] Although there have been significant advances in treatments for B-cell neoplasms, patients with refractory or relapsed disease remain without therapeutic options. In the case of relapsed ALL, the median survival rate ranges from 4 to 8 months, with less than 10% survival. in 5 years. Even more worrying is the recurrence of DLBCL, with a complete remission rate of only 7% after salvage therapy and a median overall survival of 6.3 months (RAUT; CHAKRABARTI, 2014).

[0008] Genetically modified T-cell therapy with a chimeric antigen receptor (CAR) is an innovative and revolutionary immunotherapy approach for cancer treatment, in which a patient's T cells are isolated, genetically modified, expanded, and then infused back into the patient, where they continue to expand and destroy cancer cells. These studies began in 1989, when Zelig Eshhar, an Israeli immunologist at The Weizmann Institute of Science, created the modified recombinant receptor molecule known as a chimeric antigen receptor (CAR). This was the proof-of-concept that enabled the generation of patient-derived genetically modified T cells that target a specific antigen, independent of the MHC-TCR interaction. In the 1990s, researchers began using these genetically modified T cells to fight cancer.

[0009] CAR specificity derives from the extracellular domain, derived from the antigen-binding site. A single-chain variable fragment (scFv) is generated by linking the variable region of the heavy and light chains of an immunoglobulin. A flexible peptide linker is included, and this extracellular domain is fused to an intracellular domain via a transmembrane sequence. The intracellular domain is designed to recapitulate the normal series of events by which T cells are activated. Furthermore, the incorporation of costimulatory signals into the CAR makes CAR-T cells less susceptible to downregulation by host cells, which occurs in normal immune responses against cancer. Therefore, a CAR construct reprograms T cells into “killer cells” by targeting a specific cancer antigen, and these cells can persist in the patient's body for years, aiding in immune surveillance.

[0010] The process of modifying the intracellular domain to increase efficacy led to the development of first-, second-, third-, and fourth-generation CARs. First-generation CARs consist solely of the CD3-zeta chain domain of the TCR complex and antigen recognition domains. The use of first-generation CARs was explored in clinical trials involving patients with various types of cancer. However, these studies showed only modest efficacy, primarily due to insufficient T-cell persistence in vivo. Subsequently, second-generation CARs were developed, incorporating costimulatory domains such as CD28 or 4-1BB (CD137), increasing CAR-T cell survival and proliferation and ultimately leading to improved antitumor efficacy. Third-generation CARs combine the CD3-zeta domain with more than two costimulatory domains.Finally, fourth-generation CARs incorporate additional genes to enhance the antitumor activity of CAR-T cells. Recently, other genetic modifications (i.e., receptor design or different vectors used for gene delivery) have been evaluated in preclinical studies (in vitro or in vivo biological tests) and clinical trials.

[0011] In 2017, the FDA approved CAR-T cell therapies, heralding a new era in cancer treatment, such as acute lymphoblastic leukemia (ALL) relapsed or unresponsive to conventional therapy, and also for relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other rare B-cell lymphomas.

[0012] Building on this initial success, CAR-T cell therapies are now being developed and tested for other cancers.

[0013] US2018118845 describes a chimeric receptor, wherein the cytoplasmic domain comprises a portion of the 4-1BB signaling domain. SEQ ID NO: 20 of said invention comprises SEQ ID NO: 3 (CD8 transmembrane domain), SEQ ID NO: 5 (co- stimulatory domain 41BB) and SEQ ID NO: 7 (intracellular T-cell signaling domain of CD3ζ). However, the scFV region used is derived from clone FMC63. Picanço-Castro, V. et al. (2020) developed a platform to generate and expand CAR-T cells, based on a lentiviral vector composed of a second-generation CAR, with the 41BB and CD3-ζ domains. However, the scFv domain of the anti-CD19 CAR construct of the present article is different from the construct of the present application, as it is derived from clone FMC 63.

[0014] The present invention relates to an anti-CD19 chimeric antigen receptor (CAR) and aims to cure B-cell neoplasms, including, but not limited to, hematologic malignancies. The solution found (object of the invention) is the modification of immune system cells to express the anti-CD19 CAR so that, when reintroduced into the patient, they can locate and eliminate tumor cells expressing the CD19 protein on their surface.

[0015] The present invention is an adoptive cell transfer strategy of T cells transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cellular immune activity.

[0016] This application presents nucleic acid molecules encoding anti-CD19 CAR, based on the anti-CD19 antibody, clone HD37. This cellular product (anti-CD19 CAR-T) can be used to eliminate B cells in patients with leukemia, lymphoma or autoimmune diseases.

[0017] To obtain the CAR-T cell product it is necessary Genetically modify T cells to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs). CARs are antigen receptors designed to recognize cell surface antigens independently of human leukocyte antigen.

[0018] In most cancers, tumor-specific antigens are not yet well defined, but in B-cell malignancies, CD19 is an attractive tumor target. CD19 expression is restricted to both normal and malignant B cells (Uckun, et al. Blood, 1988, 71:13-29), so CD19 is a widely accepted target for safely testing CARs. Although CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been the limited in vivo expansion of CAR+ T cells, rapid cell disappearance after infusion, and disappointing clinical activity (Jena, et al. Blood, 2010, 116:1035-1044; Uckun, et al. Blood, 1988, 71:13-29).

[0019] Therefore, there is an urgent need for cancer treatment compositions and methods using CARs that can expand in vivo. The present invention addresses this need. This treatment is potentially curative for cases in which current therapies fail. Furthermore, this approach is superior to conventional therapies. SUMMARY OF THE INVENTION

[0020] In one aspect, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 scFV antigen-binding domain, a CD8 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3ζ T cell intracellular signaling domain. In another embodiment, the present invention relates to a nucleic acid molecule comprising: (i) the nucleotide sequence encoding the scFV anti-CD19 antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 15, CDR 2 as defined by SEQ ID NO: 16 and CDR 3 as defined by SEQ ID NO: 17; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 18, CDR 2 as defined by SEQ ID NO: 19 and CDR 3 as defined by SEQ ID NO: 20; (ii) the nucleotide sequence encoding the CD8 transmembrane domain comprising SEQ ID NO: 4 or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 3; and (iii) the nucleotide sequence encoding the 4-1BB costimulatory signaling region comprising SEQ ID NO: 6, or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 5;and (iv) the nucleotide sequence encoding the CD3ζ T cell intracellular signaling domain comprising SEQ ID NO: 8 or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 7.;

[0021] In another embodiment, the nucleotide sequence encoding the scFV anti-CD19 antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: 26 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 24; and the light chain variable region as defined by SEQ ID NO: 25 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 23. In another embodiment, the scFV anti-CD19 antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region. In another embodiment, the linker consists of the sequence as defined in SEQ ID NO: 21, or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 22. In another embodiment, the nucleotide sequence encoding the scFV anti-CD19 antigen-binding domain comprises SEQ ID NO: 2 or a degenerate sequence thereof, which encodes an amino acid sequence as defined in SEQ ID NO: 1. In another embodiment, the nucleic acid molecule is for the treatment of cancer. In another embodiment, the cancer is leukemias or lymphomas. In another embodiment, the diseases to be treated are non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[0022] In one embodiment, the polypeptide comprises: (i) an anti-CD19 scFV antigen-binding domain comprising a sequence with at least 90% identity to SEQ ID NO: 1; (ii) a CD8 transmembrane domain comprising a sequence with at least 90% identity to SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region comprising a sequence with at least 90% identity to SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain comprising a sequence with at least 90% identity to SEQ ID NO: 7.

[0023] In another aspect, the present invention relates to a polypeptide comprising: (i) an anti-CD19 scFV antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 9, CDR 2 as defined by SEQ ID NO: 10 and CDR 3 as defined by SEQ ID NO: 11; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 12, CDR 2 as defined by SEQ ID NO: 13 and CDR 3 as defined by SEQ ID NO: 14; (ii) a CD8 transmembrane domain; (iii) a 4-1BB costimulatory signaling region; and (iv) a CD3ζ T cell intracellular signaling domain.

[0024] In one embodiment, the anti-CD19 scFV antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: 24; and the light chain variable region as defined by SEQ ID NO: 23.

[0025] In one embodiment, the polypeptide comprises: (i) an anti-CD19 scFV antigen-binding domain, comprising a sequence as defined by SEQ ID NO: 1; (ii) a CD8 transmembrane domain, comprising a sequence as defined by SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region, comprising a sequence as defined by SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain, comprising a sequence as defined by SEQ ID NO: 7.

[0026] In another aspect, the present invention relates to a vector comprising a nucleic acid molecule as previously defined. In one embodiment, the vector is lentiviral.

[0027] In another aspect, the present invention relates to a cell, which expresses the polypeptide as previously defined.

[0028] In another aspect, the present invention relates to an in vitro method for obtaining a cell, comprising the following steps: (a) introducing nucleic acid molecules previously defined or the vector as previously defined into cells; and (b) culturing said cell under growth conditions.

[0029] In another aspect, the present invention relates to the pharmaceutical composition comprising the nucleic acid molecule, vector, polypeptides or cells, as defined above, and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present invention relates to the use of the nucleic acid molecule, vector, polypeptide, or composition, as defined above, for the treatment of cancer. In one embodiment, it is for the treatment of leukemia or lymphoma. In another embodiment, the diseases to be treated are non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0031] In another aspect, the present invention relates to the use of the construct, vector, polypeptide, or composition as defined above for the production of a medicament for the treatment of cancer. In one embodiment, it is for the treatment of leukemia or lymphoma. In another embodiment, the diseases to be treated are non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0032] In another aspect, the present invention relates to a method for treating cancer in a patient, comprising administering to the patient the nucleic acid molecule, vector, polypeptide, or composition, as previously defined. BRIEF DESCRIPTION OF THE FIGURES

[0033] The objective of the invention, together with additional advantages thereof, may be better understood by reference to the attached figures and the following descriptions: Figure 1: Structure of the CAR construction of the present invention. The arrow indicates the beginning of the translation.

[0034] Figure 2: Generation and characterization of Raji cells expressing the bioluminescent reporter enzyme luciferase. (A) Images demonstrating the relative bioluminescence intensity in culture wells containing different numbers of Raji cells expressing luciferase (Raji / Luc). (B) Graph representing the bioluminescence intensity emitted by different numbers of Raji / luc cells. (C) CD19 expression assessed by flow cytometry. The dashed histogram represents the fluorescence of Rajiluc cells labeled with the isotype control antibody.

[0035] Figure 3: In vivo antineoplastic activity of CAR-T lymphocytes. (A) Bioluminescent signal of mice after intravenous administration of Rajiluc cells. The red “X” indicates that the animals had died before the bioluminescence reading. (B) Quantification of bioluminescence demonstrating the eradication of tumor burden to levels below the lower detection limit of the equipment (~1x106 photons / s). (C) Survival curve corroborating the therapeutic effect of CAR-T cells (n = 6-10, logrank test).

[0036] Figure 4: Inclusion of the IRES+GFP fragment to facilitate in vivo tracking of CAR-T cells. Using conventional molecular cloning, the IRES+GFP fragment was cloned immediately after the CAR coding region. The IRES region allows the GFP gene to be transcribed and translated independently of the CAR. Therefore, transduction with this vector results in the concomitant expression of the anti-CD19 CAR on the cell surface and the GFP reporter protein intracellularly.

[0037] Figure 5: In vitro cytotoxicity of CAR19 / GFP T cells. (A) Images of culture wells demonstrating the bioluminescence emitted by tumor cells 0h and 24h after the start of co-cultivation. (B) Graph demonstrating the variation in bioluminescence after 24h of co-cultivation. ****p<0.0001, one-way ANOVA and Tukey's post-hoc test; n = 3.

[0038] Figure 6: In vivo antineoplastic potential of CAR-T / GFP cells. (A) Images demonstrating the bioluminescence emitted by RAJI-luc lymphoma cells. (B) Quantification of bioluminescence. *p<0.05; **p<0.01; T-test; n = 6-7. (C) Survival curves over 29 days after treatment.

[0039] Figure 7: Detection of CAR-T / GFP cells in blood, spleen, and bone marrow after treatment. (A) Representative dotplot demonstrating the identification of CAR-T / GFP cells by flow cytometry (cells positive for human CD3 and GFP). (B) Frequency of CAR-T / GFP cells in peripheral blood throughout treatment. (C) Frequency of CAR-T / GFP cells in blood, spleen, and bone marrow after euthanasia of the animals. DETAILED DESCRIPTION OF THE INVENTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the invention belongs. Conventional molecular biology techniques are well known to one skilled in the art and can be found, for example, in Ausubel et al., eds. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. NY (1987-2008), including all supplements; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989); Oligonucleotide Synthesis (M.J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R.I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds.Harwood Academic Publishers, 1995); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984; Animal Cell Culture (R.I. Freshney, ed. (1986; Immobilized Cells and Enzymes (l.R.L. Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.) .

[0041] Although the present invention may be susceptible to different embodiments, a preferred embodiment is shown in the drawings and in the following detailed discussion, with the understanding that the present description should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been illustrated and described herein. DEFINITIONS

[0042] The term “Chimeric Antigen Receptor” (CAR), as used herein, refers to an artificially constructed hybrid protein or polypeptide containing the “antigen-binding domains” The “antigen” of an antibody (e.g., a single-chain variable fragment (scFv) or nanobodies) linked to the T-cell signaling domains. The characteristics of CARs include their ability to redirect T-cell specificity and reactivity to a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition provides CARs expressed on T cells with the ability to recognize antigen independent of antigen processing, thus bypassing an important tumor escape mechanism. Furthermore, when expressed on T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T-cell receptor (TCR). The CARs of the invention have CD19 antigen specificity.

[0043] The present invention relates to the use of genetically modified T cells to stably express a desired CAR. T cells expressing a CAR are referred to herein as CAR-T cells or CAR-modified T cells. Preferably, the cell can be genetically modified to stably express an antibody-binding domain on its surface, conferring novel antigen specificity that is MHC-independent. In some cases, the T cell is genetically modified to stably express a CAR that combines an antigen-recognition domain of a specific antibody with an intracellular domain of the CD3-zeta chain or FcγRI protein in a single chimeric protein.

[0044] The CAR of the invention comprises an antigen-binding domain, a transmembrane domain, a costimulatory region and a T-cell signaling domain.

[0045] The term “antigen-binding domain,” as used herein, refers to a region that binds to a B lymphocyte antigen, such as a CD19-binding region, and is responsible for the recognition of the antigen. CD19 can be wild-type CD19 or mutant CD19. It also optionally contains a signal peptide (PS) so that the CAR can be glycosylated and anchored to the cell membrane of the effector immune cell. The CD19 antigen-binding domain is an antibody or an antigen-binding fragment thereof. The antigen-binding fragment is a Fab or scFv.

[0046] The "transmembrane domain," as used herein, connects the extracellular domain to the intracellular domain and resides within the cell membrane when expressed by a cell. A transmembrane domain may include, but is not limited to, CD8. The term "costimulatory signaling region," as used herein, refers to a protein region that stimulates CAR T cells to become less susceptible to downregulation by host cells, which occurs in normal immune responses against cancer. A costimulatory signaling region may include, but is not limited to, 4-1BB.

[0047] The term "T cell intracellular signaling domain," as used herein, refers to the series of events by which T cells are activated. A T cell intracellular signaling domain may include, but is not limited to, CD3ζ.

[0048] The term "linker" refers to a molecule or group of molecules that connect two compounds, such as two polypeptides. The linker may consist of a single linker molecule or may comprise a linker molecule and a spacer molecule, designed to separate the linker molecule and a compound by a specific distance.

[0049] “Polypeptides” as defined herein are amino acid or peptide sequences that may have 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0050] The term “vector” refers to vectors into which a nucleic acid molecule of the present invention is inserted. Vectors derived from Retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow for the stable long-term integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0051] CAR T cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example, a CAR comprising anti-CD19, CD8α hinge and transmembrane domain, and human 4-1BB and CD3zeta signaling domains.

[0052] Expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector. Vectors can be suitable for expression in human cells and can be viral or non-viral. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0053] Additionally, the expression vector can be delivered to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in other virology and molecular biology textbooks. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, a suitable vector contains a functional origin of replication in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and US Patent No. 6,326,193). Non-viral methods such as transfection of nucleic acids (mRNA, episomes, and plasmids) by electroporation, nucleofection, lipid-based transfection reagents, nanoparticles; transfection methods based on transposons or retrotransposons; CRISPR-Cas9 and TALENS.

[0054] A "lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. They can deliver a significant amount of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are examples of lentiviruses. Lentivirus-derived vectors offer the means to achieve significant levels of gene transfer in vivo.

[0055] The term "cells" refers to several types of immune cells that can be used in CAR cell therapy, including alpha-beta T cells, gamma-delta T cells, and natural killer cells. The cells can be obtained from various sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. The cells can be obtained from a unit of blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ separation. Cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, natural killer cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.Cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.

[0056] The CAR T cells of the invention are capable of replicating in vivo, resulting in long-term persistence that may lead to sustained tumor control.

[0057] The term "pharmaceutically acceptable carriers" described herein, for example, refers to vehicles, adjuvants, excipients, and diluents that are well known to those skilled in the art and readily available to the public. It is preferable that the pharmaceutically acceptable carrier be one that is chemically inert with respect to the active agents and that has no harmful side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the CAR material of the particular invention, as well as the particular method used to administer the CAR material of the invention.Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, fillers / vehicles, solvents / co-solvents, preservatives, antioxidants, suspending agents, surfactants, anti-foaming agents, buffering agents, chelating agents, sweeteners, binders, extenders, disintegrants, diluents, lubricants, fillers, wetting agents, glidants, and combinations thereof. Formulations suitable for the present invention may generally be in any physical form known in the art suitable for therapeutic use. Non-limiting examples are capsules, liquids, suspensions, gels, pastes, injectables, or implantable sustained-release formulations. Additionally, the formulations of the present invention may be administered by any route of administration known in the art, including, but not limited to, intravenous, intratumoral, intraventricular, intramuscular, intraperitoneal, intrathecal, among others.Consequently, there is a wide variety of suitable formulations of the pharmaceutical composition of the invention. Preservatives may be used. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives may optionally be used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0058] "Treating" a disease, as used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The invention relates to the administration of a genetically modified T cell expressing a CAR for the treatment of a patient with cancer or at risk of cancer using lymphocyte infusion. Preferably, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs (peripheral blood mononuclear cells) are collected from a patient in need of treatment, and the T cells are activated and expanded using methods described herein and known in the art, and then infused back into the patient.

[0059] The term "cancer," as used herein, is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Hematological cancers can be treated using the CD 19 CAR disclosed herein. Examples of hematologic cancers that can be treated using the methods of the disclosure include non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). For example, a patient may receive isolated CD19 CAR-T cells to treat non-Hodgkin lymphoma (NHL), B-chronic lymphocytic leukemia (B-CLL), or B-acute lymphocytic leukemia (ALL).In addition to hematologic cancers expressing CD19, this invention can be used to treat patients with autoimmune diseases.

[0060] The term "autoimmune disease" as used herein is defined as a disease that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others. NUCLEIC ACID CONSTRUCTION

[0061] In one aspect, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 scFV antigen-binding domain, a CD8 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3ζ T cell intracellular signaling domain.In another embodiment, the present invention relates to a nucleic acid molecule comprising: (i) the nucleotide sequence encoding the anti-CD19 scFV antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 15, CDR 2 as defined by SEQ ID NO: 16 and CDR 3 as defined by SEQ ID NO: 17; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 18, CDR 2 as defined by SEQ ID NO: 19 and CDR 3 as defined by SEQ ID NO: 20; (ii) the nucleotide sequence encoding the CD8 transmembrane domain comprising SEQ ID NO: 4 or a sequence. degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 3; and (iii) the nucleotide sequence encoding the 4-1BB costimulatory signaling region comprising SEQ ID NO: 6, or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 5; and (iv) the nucleotide sequence encoding the CD3ζ T cell intracellular signaling domain comprising SEQ ID NO: 8 or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 7.

[0062] In another embodiment, the nucleotide sequence encoding the scFV anti-CD19 antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: 26 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 24; and the light chain variable region as defined by SEQ ID NO: 25 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 23.

[0063] In another embodiment, the anti-CD19 scFV antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region.

[0064] In another embodiment, the linker consists of the sequence as defined in SEQ ID NO: 21, or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 22.

[0065] In another embodiment, the nucleotide sequence encoding the scFV anti-CD19 antigen-binding domain comprises SEQ ID NO: 2 or a degenerate sequence thereof, which encodes an amino acid sequence as defined in SEQ ID NO: 1.

[0066] In another embodiment, the nucleic acid molecule is for the treatment of cancer.

[0067] In another embodiment, cancers are leukemias or lymphomas.

[0068] In another embodiment, the diseases to be treated are non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[0069] The present invention describes an anti-CD19 CAR construct. The isolated nucleic acid sequences encode a chimeric antigen receptor (CAR), in which the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain. This CAR can be used alone in an expression vector and / or mRNA (2nd generation CAR) or co-expressed with secreted proteins such as interleukins (4th generation CAR).

[0070] Below are the domains present in the CAR construct of the present invention Table 1A: Domains comprised in the CAR construct of the present invention #1 CAR.19-41BB-CD3z QI TK GV eia . R2: O: EQ A AC CCGTCAACC SEQ ID NO: 3 CD8 transmembrane domain (amino acids) VI G TG CT A GT ETGAG AC 1) [ Anti-CD19 AR comprising CD3-zeta and the 4-1BB costimulatory domain (also referred to as CART19 T cells). The CART19 T cells of the invention can undergo robust T cell expansion in vivo and can establish CD19-specific memory cells that persist at high levels for a long period in the blood and bone marrow. In some cases, CART19 T cells of the invention infused into a patient can eliminate acute lymphoblastic leukemia or non-Hodgkin's lymphoma (NHL) cells. However, the invention is not limited to CART19 T cells. Rather, the invention includes any antigen-binding moiety fused to one or more intracellular domains selected from the group comprising a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zeta signal domain, and any combination thereof.

[0072] The CARs disclosed in this document comprise an extracellular domain capable of binding to an antigen, at least one transmembrane domain of the tumor necrosis factor receptor superfamily, and at least one intracellular domain. The anti-CD19 CARs used in commercial products are all based on the FMC 63 antibody, while the construct of the present application is based on the HD-37 clone.

[0073] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains the antigen-binding domains of an antibody. In this case, a novel single-chain variable fragment (scFv) is linked to T-cell signaling domains by the transmembrane domain. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity to a selected target in a non-MHC-restricted manner and to exploit the antigen-binding properties of monoclonal antibodies. Unrestricted antigen recognition by MHC gives CAR-expressing T lymphocytes the ability to recognize antigen independently of antigen processing, thus providing a greater mechanism for tumor escape. Furthermore, when expressed on T lymphocytes, CARs do not advantageously dimerize with the alpha and beta chains of the endogenous T lymphocyte receptor (TCR). POLYPEPTIDE

[0074] In another aspect, the present invention relates to a polypeptide, comprising: (i) an anti-CD19 scFV antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 9, CDR 2 as defined by SEQ ID NO: 10, and CDR 3 as defined by SEQ ID NO: 11; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 12, CDR 2 as defined by SEQ ID NO: 13, and CDR 3 as defined by SEQ ID NO: 14; SEQ ID NO: 14; (ii) a CD8 transmembrane domain; (iii) a 4-1BB costimulatory signaling region; and (iv) a CD3ζ T cell intracellular signaling domain.

[0075] In one embodiment, the anti-CD19 scFV antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: 24; and the light chain variable region as defined by SEQ ID NO: 23.

[0076] In another embodiment, the polypeptide comprises: (i) an anti-CD19 scFV antigen-binding domain comprising a sequence with at least 90% identity to SEQ ID NO: 1; (ii) a CD8 transmembrane domain comprising a sequence with at least 90% identity to SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region comprising a sequence with at least 90% identity to SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain comprising a sequence with at least 90% identity to SEQ ID NO: 7.

[0077] In another embodiment, the polypeptide comprises: (i) an anti-CD19 scFV antigen-binding domain, comprising a sequence as defined by SEQ ID NO: 1; (ii) a CD8 transmembrane domain, comprising a sequence as defined by SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region, comprising a sequence as defined by SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain, comprising a sequence as defined by SEQ ID NO: 7. VECTOR

[0078] In another aspect, the present invention relates to a vector comprising the nucleic acid molecule as defined above.

[0079] In one embodiment, the vector is a lentiviral vector. CELL

[0080] In another aspect, the present invention relates to the cell expressing the polypeptide as defined above. IN VITRO METHOD

[0081] In another aspect, the present invention relates to an in vitro method for obtaining a cell, comprising the steps of (a) introducing nucleic acid molecules as defined above or with the vector as defined above; and culturing said transformed cell under growth conditions. PHARMACEUTICAL COMPOSITION

[0082] In another aspect, the present invention relates to the pharmaceutical composition comprising the nucleic acid molecule, polypeptide vector or cells as defined above and a pharmaceutically acceptable carrier.

[0083] The CAR T cells of the invention can be used in therapeutic applications. For example, they can be used as a "medicine," which, for the purposes of this invention, refers to a population of T cells genetically modified to express a CAR that can be administered to a patient.

[0084] In some embodiments, the drug may be tested for one or more contaminants, such as human viruses like HIV, HBV, HCV, CMV, mycoplasma, or bacterial endotoxins. Additionally, or alternatively, the drug may be tested for sterility.

[0085] In some embodiments, at least 20%, at least 30%, 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the cells obtained express the CAR of the invention.

[0086] In a particular embodiment, the cells of the invention are subjected to formulation steps for subsequent freezing. Once formulated, the cells are cryopreserved and can be used for therapeutic purposes, particularly in the treatment of neoplastic, inflammatory, and immune-mediated diseases, when requested by the attending physician. Additionally and / or alternatively, the formulated cells can be used in combination with other therapies. USE FOR TREATMENT

[0087] In another aspect, the present invention relates to the use of the nucleic acid molecule, vector, polypeptide or composition, as defined above, for the treatment of cancer.

[0088] In one embodiment, the use is for the treatment of leukemia or lymphoma.

[0089] In another embodiment, the use is for the treatment of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). USE FOR THE MANUFACTURE OF A MEDICATION FOR THE TREATMENT

[0090] In another aspect, the present invention relates to the use of the nucleic acid molecule, vector, polypeptide or composition, as defined above, for the production of a medicament for the treatment of cancer.

[0091] In one embodiment, the drug is for the treatment of leukemia or lymphoma.

[0092] In another embodiment, the drug is for the treatment of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). METHOD FOR TREATING CANCER

[0093] In another aspect, the present invention relates to a method for treating cancer in a patient, comprising administering to the patient the nucleic acid molecule as previously defined, the vector as previously defined, the polypeptide as previously defined, or the composition as previously defined.

[0094] A genetically modified T cell population of the present application may be administered to a patient for therapeutic purposes, for example, the treatment of a cancer that is targeted by the CAR construct expressed by the genetically modified T cell population.

[0095] A patient can be any individual for whom treatment or therapy is intended. In some embodiments, the patient is a mammal. In another embodiment, the patient is a human being.

[0096] Non-limiting examples of cancers that can be treated using a genetically modified T cell population produced by the described method include, but are not limited to, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), chronic myeloid leukemia (CML), among others.

[0097] Administration may include placement (e.g., transplantation) of the genetically modified T cell population into a patient by a method or route that results in at least partial localization of the genetically modified T cell population to the desired site, such as a tumor site, so as to produce the desired effect(s). The genetically modified T cell population may be administered by any appropriate route that results in delivery to a desired site in the patient where at least a portion of the implanted cells or cell components remain viable. The period of viability of the cells after administration to a patient may be as short as a few hours, for example, twenty-four hours, a few days, several years, or even the lifetime of the patient. For example, in some aspects described herein, an effective amount of the genetically modified T cell population may be administered by a systemic route of administration, such as intraperitoneally or intravenously.

[0098] In some embodiments, the genetically modified T cell population is administered systemically, which refers to administering a population of cells not directly to a target site, tissue, or organ, but rather to the subject's circulatory system.

[0099] Suitable routes of administration include injection, infusion, or instillation. In some embodiments, the route is intravenous.

[0100] An effective amount refers to the amount of a genetically modified T-cell population needed to prevent or alleviate at least one or more signs or symptoms of cancer and refers to a sufficient amount of a genetically modified T-cell population to provide the desired effect, e.g., to treat a patient with the disease. An effective amount also includes an amount sufficient to prevent or delay the development of a disease symptom, alter the course of a disease symptom (e.g., but not limited to, slowing the progression of a disease symptom), or reversing a disease symptom. It is understood that, for any given case, an appropriate effective amount can be determined by one skilled in the art using routine experimentation.

[0101] An effective amount of a genetically modified T cell population may include at least 10 2cells, at least 5x10 2 cells, at least 10 3 cells, at least 5x10 3 cells, at least 10 4 cells, at least 5x10 4 cells, at least 10 5 cells, at least 2x10 5 cells, at least 3x10 5 cells, at least 4x10 5 cells, at least 5x10 5 cells at least 6x10 5 cells, at least 7x10 5 cells, at least 8x10 5 cells, at least 9x10 5 cells, at least 1x10 6 cells, at least 2x10 6 cells, at least 3x10 6 cells, at least 4x10 6 cells, at least 5x10 6 cells, at least 6x10 6 cells, at least 7x10 6 cells, at least 8x10 6 cells, at least 9x10 6cells, or multiples thereof per kilogram of the patient. The efficacy of a treatment using the genetically modified T cell population produced as described in this application can be determined by a person skilled in the art.

[0102] The CAR T cell of the present invention may be combined with another therapeutic agent. This therapeutic agent may be selected from the group consisting of a radionucleotide, an immunomodulator, an immunoconjugate, a hormone, a cytokine, an enzyme, a photoactive therapeutic agent, a cytotoxic drug, a toxin, an angiogenesis inhibitor, a therapeutic antibody, an antibody-drug conjugate, etc., and combinations thereof.

[0103] A treatment is considered "effective" if one or all of the signs or symptoms of, for example, functional target levels are altered in a beneficial way, or if other symptoms or markers of cancer clinically accepted treatments are improved or mitigated. Effectiveness can also be measured by whether a patient's condition worsens, as assessed, for example, by hospitalization or the need for medical interventions (e.g., disease progression is halted or at least slowed).

[0104] The methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual and includes: (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing the regression of symptoms; and (3) preventing or reducing the likelihood of developing symptoms. Genetically modified T cell populations manufactured as described herein may also be used in combination therapies. For example, the genetically modified T cell population manufactured as described herein may be used in combination with other therapeutic agents to treat the same indication or to enhance the efficacy of the genetically modified T cell population and / or reduce the side effects of the genetically modified T cell population.

[0105] In order to elucidate the present invention, the following are presented experimental results and embodiments of the invention to demonstrate the inventive step of the present invention. EXAMPLES EXAMPLE 1 Production of anti-CD19 CAR-T

[0106] The CAR.CD19 construct (schematic representation Figure 1), is composed of: a leader sequence that is identical to the amino acid leader sequence of interleukin 2 (IL-2) with 20 amino acids (amino acids 1 to 20 of SEQ ID NO: 1), an anti-CD19 scFv derived from clone HD-37 composed containing: variable light chain (VL), binding sequence of Glycine-serine connecting the VL and VH chains (G4S) and variable heavy chain (VH). The VH and VL chains are derived from the anti-CD19 monoclonal antibody (clone HD-63). It has a spacer and transmembrane region derived from the human CD8a molecule, an intracellular costimulatory domain derived from the human 4-1BB (CD137) molecule (42 amino acids), and the intracellular domain of the human CD3zeta chain (isoform 3) (112 amino acids). T cell enrichment

[0107] Monocyte depletion aims to enrich the CD3-positive lymphocyte population. Monocyte depletion can be performed by adherence to plastic or by CD3 T cell selection or CD4 / CD8 T cell selection using magnetic columns or flow cytometry. T cells will be harvested, washed twice, and two samples of the cell suspension will be collected for cell count, viability analysis, and markers (CD45, CD14, CD3, CD19, CD56 / 16). Lymphocyte activation

[0108] Lymphocyte activation will be performed on D+1, and the goal is to achieve a percentage of cells with CD25 / 69 surface markers greater than or equal to 20%. At this stage, 2x10 6 cells / well in 48-well plates, diluted in AIM V medium supplemented with 5% AB serum, 100 IU / mL IL-2 and 3:1 (beads:cells) CTS Dynabeads® (Invitrogen, Carlsbad, CA), followed by a 24-hour incubation at 37°C and 5% CO2. After this time, T cells will be collected, washed twice and 2 samples of the cell suspension will be collected for cell counting, viability analysis and markers (CD45, CD14, CD25, CD8, CD69, CD4, CD19, CD16 / 56). Transduction

[0109] After the activation process, lymphocyte transduction will be performed. For this, T cells (1x10 6 cells / well) will be transduced in a 24-well plate with the lentivirus (MOI 5) by incubation for 30 min, 37°C followed by a 90-min centrifugation at 1258 g and 320°C in the presence of 8 mg / mL of polybrene. After centrifugation, the cells will be incubated at 37°C and 5% CO2. After 24 hours, 1.5 ml of supplemented medium, AIM V with 5% AB serum and 100 IU / ml of IL-2, will be added to each well. Four days after transduction, two samples of the cell suspension will be collected for QC assays: cell count, analysis of CD45, CD14, CD3, CD16 / 56, CD8, CD3, CD4, and CAR19 markers, and the percentage of CAR-transduced cells. Expansion

[0110] T cells will be expanded in complete medium (AIM V supplemented with 5% human AB serum and 100 U / mL IL 2) to maintain a CAR-T cell concentration of 1×10 6cells / mL and incubated at 37°C, 5% CO2. Cell proliferation will be maintained by replacing 100% of the supplemented culture medium every two days. On day 7, the activation beads are removed. Lymphocyte expansion will also be evaluated in PL-30, PL-70, Prodigy, and Sepax bags. Cell number and viability will be determined at predefined expansion periods using 7AAD / annexin-V flow cytometry, for which quality control (QC) assays will be performed. Cell expansion will be continued until the dose of CAR-T cells required for the patient's treatment is reached, with a desirable dose of ≥1x10 6 positive CAR-T cells per kg of patient body weight. EXAMPLE 2 Detection of CAR expression on the surface of CAR-T cells

[0111] The objective is to validate the gene transfer process and transgene expression by detecting CAR on the surface of CAR-T cells.

[0112] After manufacturing, CAR-T cells were incubated with the murine anti-F(ab')2 antibody conjugated with the fluorochrome Alexa 647 and the 7-AAD cell viability dye to exclude dead cells. After staining, samples were evaluated by flow cytometry to determine the frequency of viable cells expressing the CAR on the surface.

[0113] Nine batches of CART cells produced from cells from different patients were evaluated. The frequency of cells expressing CAR on the cell surface ranged from 10.2% to 50.8%, with a median of 35.9% (Table 1).

[0114] Table 1: Frequency of cells expressing CAR on the cell surface. After transduction and in vitro expansion, samples of the cell product were labeled with the murine anti-F(ab')2 antibody for CAR detection. After labeling, the frequency of CAR+ cells was T cells, a sine qua non condition to allow antigen recognition- target (CD19) on the surface of neoplastic cells. EXAMPLE 3 In vitro antitumor activity and selectivity for the CD19 target

[0116] The goal is to demonstrate that CAR-T cells are capable of eliminating tumor cells in vitro that express the CD19 target antigen and that CAR-T cells do not eliminate tumor cells that do not express the CD19 antigen on their surface. Cell lines

[0117] As cellular models expressing the target antigen CD19, we used the Raji (Burkitt's lymphoma) and SUP-B15 (B-cell acute lymphoblastic leukemia) cell lines. The K562 cell line (chronic myeloid leukemia) was used as a control that does not express the target antigen CD19. Co-cultivation method

[0118] Initially, tumor cell lines were labeled with the fluorescent dye PKH67 to allow their identification by flow cytometry. Subsequently, the tumor cells were mixed with the final cellular product (CAR-T) at a 1:5 ratio (tumor cell:cell product). The frequency of tumor cells (labeled with the PKH67 reagent) was determined at baseline and after 24 hours of co-cultivation and used to calculate the percentage of tumor lysis.

[0119] CAR-T cells were able to lyse both CD19-positive cell lines (Table 2). In all cocultures performed, CAR-T cells did not lyse cells from the K562 cell line, which does not express CD19.

[0120] These results demonstrate that T cells modified to express the CAR related to this invention have selective cytotoxic capacity against CD19-positive cells.

[0121] Table 2: Percentage of tumor cell lysis after 24h of co-cultivation with the final cell product. CD19-positive (Raji and SUP-B15) and CD19-negative (K562) tumor cells were co-cultured with the final cell product at a ratio of 1:5 (tumor cell: final cell product) for 24 hours. The percentage of lysis was determined by quantifying the number of dead tumor cells at the beginning and end of co-cultivation. N = 6 donors (patients). Percentage of lysis after 24 hours of co-cultivation In vivo antitumor activity

[0122] The objective is to demonstrate the ability of CAR-T cells to eliminate CD19+ neoplastic cells in vivo. Mouse line

[0123] To avoid the ready rejection of human cells, in vivo studies were performed with the immunodeficient mouse strain NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ, also known as NOD scid gamma (NSG™). The matrices of this line were obtained from Jackson Laboratories (lineage 005557), and the descendant animals are maintained at the Laboratory of Experimental Studies in Animals (LEEA) of the Ribeirão Preto Blood Center. Animals of the NSG line have a spontaneous loss-of-function mutation in the Prkdc gene (Prkdc scid ) and a targeted loss-of-function mutation in the interleukin 2 receptor gamma chain gene (Il2rg tm1Wjl ). As a result of the mutations, NSG animals lack functional T, B, and NK cells (“https: / / www.jax.org / strain / 005557”). Furthermore, the genetic background of this strain (NOD / ShiLtJ) contributes to the immunosuppressed phenotype by the absence of a hemolytic complement system in addition to dysfunctional dendritic cells and macrophages (SHULTZ et al., 1995). Murine model of lymphoma

[0124] The tumor model was Burkitt's lymphoma using Raji cells (ATCC - CCL-86 cell line). To allow non-invasive, horizontal, and real-time tracking of neoplastic progression, Raji cells were transduced with a lentiviral vector encoding the luminescent reporter enzyme luciferase 2. Furthermore, expression of the target antigen CD19 was confirmed by flow cytometry, and cell identity was confirmed by short tandem repeat (STR) profiling. Production of test items (CAR-T cells)

[0125] After signing the Informed Consent Forms, we collected the leukoreduction systems used in the plateletpheresis procedure from three healthy donors. The leukoreduction systems were washed with phosphate-buffered saline to collect the retained cells. After washing, the mononuclear cells were isolated by Ficoll-Paque PLUS gradient centrifugation and transduced with lentiviruses carrying the genetic construct encoding the anti-CD19 CAR. For transduction, we used two batches of lentiviral particles (Table 3). After transduction and expansion, the CAR-T cells were cryopreserved at -80°C. After 24 hours, the tubes were transferred to liquid nitrogen containers until use in the experiments.

[0126] Table 3: Characteristics of the lentivirus batches produced. Characterization of lentiviral particles Donor Lentivirus batch Viral titer Mycoplasma a Endotoxin bTest used (IU / mL) (EU / mL) microbiological a b . EXAMPLE 5 Treatment and monitoring of tumor burden

[0127] Initially, 16 male immunodeficient mice of the NSG strain (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ), with 8-12 weeks of age, received an intravenous injection (lateral caudal vein) of suspensions containing 5x10 5 Raji cells luc in 200 μL of phosphate buffered solution (PBS) to establish the disseminated lymphoma model. Six days after tumor cell infusion, 6 of the animals were treated with suspensions containing 5x10 6anti-CD19 CAR T cells in 200 μL of PBS. The remaining 10 animals received only PBS (vehicle) and served as experimental controls. Tumor progression was monitored by quantifying bioluminescence using the IVIS Lumina System. Before each reading, the animals received an injection of the substrate D-luciferin (150 mg / kg) intraperitoneally. Readings were performed 3 days before treatment (D-3), on the day of treatment (D0), and on days D+3, D+7, D+10, and D+15. Animals that presented ≥20% body weight loss or hindlimb paralysis were euthanized by administration of anesthetic (ketamine and xylazine). Generation and characterization of luciferase-expressing Raji cells (Raji luc ):

[0128] To confirm the cell line identity, genomic DNA samples from Raji cells were sent for short tandem repeat (STR) analysis to the laboratory of Prof. Aguinaldo Luis Simões, from the Genetics Department of the Ribeirão Preto School of Medicine. The test consists of PCR amplification of eight different hypervariable regions containing short tandem repeats. In this test, the PCR reaction indicates how many repetitions of a specific DNA motif (e.g., ATGG) occur in a given allele. For example, a value of 10.12 for the CSF1PO locus indicates that the DNA motif present in this region is repeated 10 times in one allele and 12 times in the other. The short tandem repeat profile for the eight regions tested was identical to the profile reported for Raji cells available in the ATCC repository (https: / / www.atcc.org / ) (Table 4). These data confirm the identity of the Raji cell line used in the study described here.

[0129] Table 4: Short tandem repeat profile of 8 different loci of the genome. Data from the Raji line used in our study (column 2) were compared to data made available by the ATCC repository (column 3). Number of repeats of motifs Locus [ were transduced with lentiviral particles carrying the genes encoding the bioluminescent enzyme luciferase and the enzyme puromycin N-acetyltransferase, which confers resistance to the antibiotic puromycin. After transduction using 8 viruses / cell, the cells were cultured for 6 days in the presence of 2 μg / mL puromycin to eliminate non-transduced cells. At the end of this period, Raji cells, now expressing luciferase (Raji luc), were incubated with 150 μg / mL of the substrate D-luciferin and their bioluminescence was quantified using the IVIS Lumina System and Living Image 3.0 software (Perkin Elmer). These analyses demonstrated that the bioluminescent signal was proportional to the number of Raji cells luc (figure 2A-B) and the average bioluminescence intensity was 1.16x10 3 ± 0.13x10 3 photons / second / cell (n = 4). Finally, virtually all Raji cells luc (99.7%) were positive for CD19, as assessed by flow cytometry (Figure 2C). EXAMPLE 6 Characterization of the test item (CAR-T cells)

[0131] CAR-T cells were evaluated for their in vivo potency in a lymphoma model. Table 5 summarizes the characteristics of the products and the animals that received them.

[0132] Table 5: Information about CAR T cells produced for the preclinical trial (Creative Biolabs vector). Sample ID DN2 PBMC#11 PMBC#12 AB L % Effect of CAR-T cells on tumor progression and animal survival

[0133] Three days after treatment with CAR-T cells, five of the six animals showed complete remission of all lymphoma foci (Figure 3A). Only one of the treated animals showed partial regression of the lymphoma with subsequent relapse (Figure 3A, fourth animal in the CAR-T group). In the animals that received only PBS, there was uninterrupted progression of the lymphoma, affecting the entire body (Figure 3A). Twenty-one days after tumor cell infusion, all animals in this group had already been euthanized due to hind limb paralysis caused by lymphoma cell infiltration or loss of at least 20% of their initial body weight. In contrast, treatment with CAR-T cells ensured survival of five of the six animals for up to 36 days after treatment, when the experiment was stopped (Figure 3C). EXAMPLE 8 Persistence and distribution of CAR-T cells in peripheral blood and major hematopoietic organs

[0134] The objective is to demonstrate the kinetics and biodistribution of CAR-T cells in vivo. Inclusion of the GFP (green fluorescent protein) reporter gene to facilitate the tracking of CAR-T cells in vivo.

[0135] To investigate the persistence and biodistribution of CAR-T cells in vivo, the lentiviral vector was modified by including the gene encoding green fluorescent protein (GFP) and the internal ribosome entry site (IRES) downstream of the anti-CD19 CAR gene (Figure 4). The IRES region allows the GFP gene to be transcribed and transduced independently of the CAR. Therefore, transduction with this vector results in the concomitant expression of the anti-CD19 CAR on the cell surface and the GFP reporter protein intracellularly. Generation of CAR-T / GFP cells

[0136] After generation of the lentiviral vector and lentiviral particles, CAR-T cells were transduced (5 viruses / cell) in the presence of 8 µg / mL polybrene and evaluated for CAR and GFP expression by flow cytometry. CAR-T / GFP cells were also evaluated for their antitumor potential in vitro and in vivo (described below). Evaluation of the antineoplastic potential of CAR-T / GFP cells in vitro

[0137] CAR-T / GFP cells were co-cultured with Raji (CD19+) or K562 (CD19-) cell lines previously modified to express luciferase (referred to as Raji luc and K562 luc , respectively). Bioluminescence quantification was used to determine tumor cell death. For co-cultivation, RAJI cells luc or K562 luc they were mixed 1:1 with unmodified T cells or CAR-T / GFP cells. Tumor cells cultured alone served as additional controls for the experiment. Bioluminescence was quantified immediately after plating and 24 hours after co-cultivation. The variation in bioluminescence between these two time points was used to estimate tumor cell death. Assessment of the antineoplastic potential of CAR-T / GFP cells in vivo:

[0138] To evaluate the antineoplastic potential in vivo, 13 male NOD-scid gamma (NSG) immunodeficient mice aged 10-12 weeks received 5x10 5 RAJI cells luc intravenously (lateral caudal vein). Six days after injection of tumor cells, the animals were randomly distributed into two groups: one group (n = 7) received 5x10 6CAR-T / GFP cells intravenously, and the control group (n = 6) received only phosphate-buffered saline (PBS). After treatment, tumor burden was monitored by bioluminescence quantification, and peripheral blood samples were taken periodically to assess the presence of circulating CAR-T / GFP cells. Sample processing for CAR-T / GFP cell quantification by flow cytometry

[0139] On days 2, 7, 23, and 29 after treatment, 50 μL of blood was collected from the submandibular vein of the animals to evaluate the presence of circulating CAR-T / GFP cells by flow cytometry. For flow cytometry analysis, whole blood samples were subjected to erythrocyte lysis in ACK buffer. The samples were then washed and incubated with anti-human CD19 (to exclude circulating tumor cells) and anti-human CD3 (to identify human T lymphocytes) antibodies for 15 min at room temperature and analyzed by flow cytometry. After exclusion of doublets, dead cells (DAPI + ) and circulating lymphoma cells (CD19 + ), CAR-T / GFP cells were identified as GFP + CD3 + and quantified. In vitro antineoplastic potential of CAR-T / GFP cells

[0140] After 24 hours of co-cultivation, CAR-T / GFP cells eliminated approximately 75% of RAJIluc cells and were unable to eliminate the K562luc cell line (Figure 5).

[0141] These data indicate that CAR-T / GFP cells are functional and specific for CD19 cells + . Unmodified T cells did not induce significant killing of any tumor cells, indicating that the cytotoxic effect of CAR-T / GFP cells is due to the expression of the anti-CD19 CAR. Taken together, these data demonstrate that inclusion of the GFP-encoding gene in the viral vector does not impede the cytotoxic activity of CAR-T / GFP cells. EXAMPLE 9 In vivo antineoplastic potential of CAR-T / GFP cells:

[0142] In vivo bioluminescence quantification demonstrated that CAR-T / GFP cells reduced tumor burden by up to a thousandfold (Figure 6A-B), corroborating the in vivo antineoplastic potential of CAR-T / GFP cells. This therapeutic effect led to longer survival in the treated group (p<0.0001), with only one death occurring over 29 days after treatment. Conversely, all control animals that received only PBS (vehicle) succumbed to the disease after 15 days (Figure 6C). These data demonstrate that coexpression of the GFP reporter protein does not eliminate the cytotoxic potential of CAR-T cells. EXAMPLE 10 Persistence and biodistribution of CAR-T / GFP cells in vivo:

[0143] Expression of the GFP reporter protein easily allowed identification of CAR-T / GFP cells by flow cytometry (Figure 7A). Using this methodology, it was observed that CAR-T cells persisted in the peripheral blood throughout the study at levels that were not greater than 2% of whole blood cells (Figure 7B). After 29 days of treatment, the experiment was stopped and the animals were euthanized. At this time, a terminal analysis was performed on the peripheral blood, spleen, and bone marrow of the animals to evaluate the presence of CAR-T / GFP cells by flow cytometry. CAR-T / GFP cells were identified in all tissues analyzed, at a frequency of less than 1%, and there was a tendency for accumulation in the spleen compared to peripheral blood and bone marrow (Figure 7C). These data demonstrate that the generated vector allowed the identification of small numbers of CAR-T / GFP cells in the analyzed samples and that CAR-T / GFP cells persisted in the peripheral blood and main hematopoietic organs of the animals throughout the study. EXAMPLE 11 Assessment of the chromosomal integrity of CAR-T cells by G-banding

[0144] The objective is to evaluate the chromosomal integrity of CAR-T cells.

[0145] At the end of the expansion, an aliquot of the CAR-T cells is sent to the Cytogenetics Laboratory at the Ribeirão Preto School of Medicine's Hospital das Clínicas. There, the samples are processed according to the Giemsa banding protocol, and 20 metaphases are analyzed for the numerical and structural integrity of the chromosomes. Karyotypes are considered normal when there are no structural anomalies (deletions, inversions, or translocations) and the number of chromosomes is 46, comprising 22 pairs of somatic chromosomes and one pair of sex chromosomes (XX for females and XY for males).

[0146] Samples of nine cellular products were analyzed. We observed no numerical or structural alterations within the resolution limit of the Giemsa banding technique. All karyotypes were therefore considered normal (Table 6), and no anomalies were found. numerical or structural in chromosomes.

[0147] Table 6: Summary of karyotyping results for nine cellular products. Sample Result PCCAR 1 Normal Karyotype E Integrated copy number of the transgene

[0148] The objective is to determine the number of integrated copies of the transgene in the final cellular product.

[0149] After expansion, an aliquot of the final cellular product is subjected to DNA extraction with a commercial kit (QIAamp DNA). The samples are then subjected to a droplet digital PCR (ddPCR) reaction using a probe / primer set against the LTR region of the lentiviral vector (used to quantify the transgene copy number) (SEQ ID NO: 27-29) and two other probe / primer sets against regions of the endogenous genes CDKN1A and RPP30 (used to quantify the genome or cell number). After reading the reaction, the amplification values ​​of the transgene (LTR) and the endogenous genes (CDKN1A or RPP30) are used to calculate the number of integrated transgene copies per cell.

[0150] The number of integrated copies was determined for 9 samples, 4 from healthy donors (DN) and 5 from patients (PCCAR). All samples had fewer than 2 copies of the transgene per cell (Table 7). The lower the number of integrated copies, the lower the risk of insertional mutagenesis and, therefore, the greater the safety. No There is a guide issued by ANVISA, EMA, or FDA that defines the maximum acceptable value for this test. However, a communication from FDA member Ramjay S. Vatsan delivered 7 oThe International Society for Bioprocess Technology (ISBioTech) meeting (March 7, 2017) indicates that a recommended value would be less than or equal to 5 copies of the transgene per cell. This statement is referenced in a publication by a team from the Division of Advanced Therapies of the National Institute for Biological Standards and Control (NIBSC) (DOI: 10.1089 / hgtb.2017.078). Therefore, according to these criteria, all cellular products have a low number of integrated copies of the transgene (Table 7), which is another indication of product safety.

[0151] Table 7: Number of integrated copies of the transgene (CAR vector) in the cell products. Samples from healthy donors (DN) and patient samples (PCCAR) were analyzed. Sample ID Conclusion Number copies / cell

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[0153] Jena B, Dotti G, Cooper LJ. Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood. 2010 Aug 19;116(7):1035-44. doi: 10.1182 / blood-2010-01-043737. Epub 2010 May 3. PMID: 20439624; PMCID: PMC2938125.

[0154] Picanco-Castro V, Gonçalves Pereira C, Swiech K, Ribeiro Malmegrim KC, Tadeu Covas D, Silveira Porto G. Emerging CAR T cell therapies: clinical landscape and patent technological routes. Hum Vaccin Immunother. 2020 Jun 2;16(6):1424-1433. doi: 10.1080 / 21645515.2019.1689744. Epub 2019 Dec 6. PMID: 31702480; PMCID: PMC7482707.

[0155] Raut LS, Chakrabarti PP. Management of relapsed-refractory diffuse large B cell lymphoma. South Asian J Cancer. 2014 Jan;3(1):66-70. doi: 10.4103 / 2278-330X.126531. PMID: 24665451; PMCID: PMC3961873.

[0156] Sambrook, Joseph. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, 2001.

[0157] Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B, McKenna S, Mobraaten L, Rajan TV, Greiner DL, et al. Multiple defects in innate and adaptive immunologic function in NOD / LtSz-scid mice. J Immunol. 1995 Jan 1;154(1):180-91. PMID: 7995938.

[0158] Uckun FM, Jaszcz W, Ambrus JL, Fauci AS, Gajl-Peczalska K, Song CW, Wick MR, Myers DE, Waddick K, Ledbetter JA. Detailed studies on expression and function of CD19 surface determinant by using B43 monoclonal antibody and the clinical potential of anti-CD19 immunotoxins. Blood.1988 Jan;71(1):13-29. PMID: 3257143.

Claims

1 / 6 CLAIMS 1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 scFV antigen-binding domain, a CD8 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3ζ T cell intracellular signaling domain.

2. The nucleic acid molecule of claim 1, comprising: (i) the nucleotide sequence encoding the anti-CD19 scFV antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 15, CDR 2 as defined by SEQ ID NO: 16, and CDR 3 as defined by SEQ ID NO: 17; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 18, CDR 2 as defined by SEQ ID NO: 19 and CDR 3 as defined by SEQ ID NO: 20;(ii) the nucleotide sequence encoding the CD8 transmembrane domain comprising SEQ ID NO: 4 or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 3; and (iii) the nucleotide sequence encoding the 4-1BB costimulatory signaling region comprising SEQ ID NO: 6, or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO: 5; and (iv) the nucleotide sequence encoding the CD3ζ T cell intracellular signaling domain comprising SEQ ID NO: 8 or a degenerate sequence thereof, which encodes the amino acid sequence as defined in SEQ ID NO:

7.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence encoding the; 2 / 6 scFV anti-CD19 antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: 26 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO: 24; and the light chain variable region as defined by SEQ ID NO: 25 or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO:

23.

4. Nucleic acid molecule according to any one of claims 1 to 3, characterized in that the scFV anti-CD19 antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region.

5. Nucleic acid molecule according to claim 4, characterized in that the linker consists of the sequence as defined in SEQ ID NO: 21, or degenerate sequences thereof, which encode the amino acid sequence as defined in SEQ ID NO:

22. 6.Nucleic acid molecule according to any one of claims 1 to 5, characterized in that the nucleotide sequence encoding the anti-CD19 scFV antigen-binding domain comprises SEQ ID NO: 2 or a degenerate sequence thereof, which encodes an amino acid sequence as defined in SEQ ID NO:

1.

7. Nucleic acid molecule according to any one of claims 1 to 6, characterized in that it is for the treatment of cancer.

8. Nucleic acid molecule according to claim 7, characterized in that it is for leukemias or lymphomas.

9. Nucleic acid molecule according to claim 8, characterized in that it is for non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL). 3 / 6 Hodgkin lymphoma (HL) and chronic myeloid leukemia (CML).

10. Polypeptide, characterized by the fact that it comprises: (i) an anti-CD19 scFV antigen-binding domain, comprising a sequence with at least 90% identity to SEQ ID NO: 1; (ii) a CD8 transmembrane domain, comprising a sequence with at least 90% identity to SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region, comprising a sequence with at least 90% identity to SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain, comprising a sequence with at least 90% identity to SEQ ID NO:

7. 11.The polypeptide of claim 10, comprising: (i) an anti-CD19 scFV antigen-binding domain comprising a heavy chain variable region comprising CDR 1 as defined by SEQ ID NO: 9, CDR 2 as defined by SEQ ID NO: 10, and CDR 3 as defined by SEQ ID NO: 11; and a light chain variable region comprising CDR 1 as defined by SEQ ID NO: 12, CDR 2 as defined by SEQ ID NO: 13, and CDR 3 as defined by SEQ ID NO: 14; (ii) a CD8 transmembrane domain; (iii) a 4-1BB costimulatory signaling region; and (iv) a CD3ζ T cell intracellular signaling domain.

12. The polypeptide of claim 10 or 11, wherein the anti-CD19 scFV antigen-binding domain comprises the heavy chain variable region as defined by SEQ ID NO: S; and the light chain variable region as defined by SEQ ID. 4 / 6 NO: T.

13. Polypeptide according to any one of claims 10 to 12, characterized in that it comprises: (i) an anti-CD19 scFV antigen-binding domain, comprising a sequence as defined by SEQ ID NO: 1; (ii) a CD8 transmembrane domain, comprising a sequence as defined by SEQ ID NO: 3; (iii) a 4-1BB costimulatory signaling region, comprising a sequence as defined by SEQ ID NO: 5; and (iv) a CD3ζ T cell intracellular signaling domain, comprising a sequence as defined by SEQ ID NO:

7.

14. Vector, characterized in that it comprises a nucleic acid molecule as defined in any one of claims 1 to 9.

15. Vector according to claim 10, characterized in that it is a lentiviral vector.

16. A cell expressing the polypeptide as defined in any one of claims 10 to 13. 17.In vitro method for obtaining a cell, characterized by the fact that it comprises the following steps: (a) introducing nucleic acid molecules as defined in any one of claims 1 to 9 or the vector as defined in claim 14 or 15 into cells; and (b) culturing said cell under growth conditions.

18. Pharmaceutical composition, characterized by the fact that it comprises: (i) nucleic acid molecule as defined in any one of claims 1 to 9; or (ii) vector as defined in claim 14 or 15; or. 5 / 6 (iii) polypeptide as defined in any one of claims 10 to 13; or (iv) cell as defined in claim 16; and (v) a pharmaceutically acceptable carrier.

19. Use of the nucleic acid molecule as defined in any one of claims 1 to 9, of the vector as defined in claim 14 or 15, of the polypeptide as defined in any one of claims 10 to 13, or of the composition as defined in claim 18, characterized in that it is for the treatment of cancer.

20. Use according to claim 19, characterized in that it is for the treatment of leukemia or lymphoma. 21.Use according to claim 19 or 20, characterized in that it is for non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL) and chronic myeloid leukemia (CML).

22. Use of the nucleic acid molecule as defined in any one of claims 1 to 9, of the vector as defined in claim 14 or 15, of the polypeptide as defined in any one of claims 10 to 13, or of the composition as defined in claim 18, characterized in that it is for the production of a medicament for the treatment of cancer.

23. Use according to claim 22, characterized in that it is for the treatment of leukemia or lymphoma. 24.Use according to claim 22 or 23, characterized in that it is for non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B-chronic lymphocytic leukemia (B-CLL), B-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), T-acute lymphocytic leukemia (ALL), leukemia. 6 / 6 acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

25. A method for treating cancer in a patient, comprising administering to the patient the nucleic acid molecule as defined in any one of claims 1 to 9, the vector as defined in claim 14 or 15, the polypeptide as defined in any one of claims 10 to 13, or the composition as defined in claim 18.

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