Chimeric antigen receptor and car-t cells that bind bcma

Abstract

To provide pharmaceuticals suitable for treating diseases associated with pathogenic B cells.SOLUTION: The invention relates to an isolated chimeric antigen receptor polypeptide (CAR). The CAR comprises an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a B Cell Maturation Antigen (BCMA) polypeptide. The CAR preferably binds an epitope comprising one or more amino acids of residues 13 to 32 of the N-terminus of human BCMA. The invention further relates to a nucleic acid molecule encoding the CAR of the invention, a genetically modified immune cell, preferably a T cell, expressing the CAR of the invention and the use of said cell in the treatment of a medical disorder associated with the presence of pathogenic B cells, such as a disease of plasma cells, and / or memory B cells, and / or mature B cells, in particular multiple myeloma, non-Hodgkin's lymphoma or autoantibody- dependent autoimmune diseases.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an isolated chimeric antigen receptor (CAR) polypeptide comprising an extracellular antigen binding domain comprising an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide. The CAR preferentially binds to an epitope comprising one or more amino acids from residues 13 to 32 of the N-terminus of human BCMA. The present invention further relates to nucleic acid molecules encoding the CAR of the invention, genetically modified immune cells (preferably T cells) expressing the CAR of the invention, and the use of the cells in the treatment of medical disorders associated with the presence of pathogenic B cells (e.g., disorders of plasma cells, and / or memory B cells, and / or mature B cells, particularly multiple myeloma, non-Hodgkin's lymphoma, autoantibody-dependent autoimmune diseases). [Background technology]

[0002] In cancer immunotherapy, adoptive transfer (ATT) of T cells genetically modified to recognize tumor-specific or tumor-associated antigens is a promising approach to eradicate tumors and tumor stem cells, which may avoid tumor recurrence, unlike traditional chemotherapy, radiotherapy, and surgery. Furthermore, although tumors can often be controlled very well by novel pathway-selective drugs, the course of the disease usually switches to a chronic phase without clear tumor elimination.

[0003] With the advent of genetically modified T cells expressing CAR, the treatment of B-cell lymphoma / leukemia has proven to be very successful, despite the fact that patients are heavily pretreated and have previously undergone several chemotherapy and antibody therapies and even autologous / allogeneic bone marrow transplants. For example, ATT using CAR-T cells has been successfully used as a salvage therapy.

[0004] CARs are synthetically engineered immunoglobulin-derived receptors that can recognize surface antigens independently of MHC. Unlike TCRs, CARs have a broader range of affinities and can bind to target antigens without necessarily showing cross-reactivity. The target antigen must be deposited on the surface and may include tumor-associated proteins or carbohydrates or even glycolipids. Another advantage of CAR-T cells is that they can be rapidly generated by transduction of autologous T cells. Autologous T cells are CD4+ T cells that express CD4+ T cells. + or CD8 + CARs can be produced “off the shelf” and their target is typically CD19. + Widely expressed in certain tumors (>90%), as shown in B-cell leukemias and lymphomas, it has been suggested that CAR-T cells could act as a "living drug" that can be sustained after even a single T-cell infusion.

[0005] There is a strong need in medicine for chimeric antigen receptor (CAR)-T cell products as described herein. First, multiple myeloma is an incurable B-cell non-Hodgkin's lymphoma (B-NHL) derived from a malignantly transformed plasma cell clone. A particularity is that the tumor cells are mainly localized in the bone marrow. The disease is the most frequent tumor of bone and bone marrow, with a 50% 10-year survival rate in young patients who receive intensive treatment, and accounts for 2% of annual cancer deaths. The incidence rate is 5 / 100,000, with a median age at diagnosis of 70 years. This indicates that intensive long-term chemotherapy is not possible in many patients due to the presence of comorbidities. Standard treatment is chemotherapy alone or in combination with autologous stem cell transplantation, immunomodulatory drugs, localized irradiation, proteasome inhibitors, and in a very small number of patients, allogeneic stem cell transplantation can be performed. Despite the above modes of intensive treatment, the disease usually relapses and develops a second resistance after multiple treatments.

[0006] Second, a much larger group of classical B-NHL includes a variety of tumors derived from B-lymphocytes that usually home to secondary lymphoid organs (e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and subgroups of chronic lymphocytic leukemia (CLL)). The total incidence of all NHL is about 10-12 / 100,000 (>85% of B-cell origin), but the majority of cases are adult disease, with a large increase in older people. Demographic trends predict that the total number will increase due to the aging of Western societies. Clinically, B-NHL consists of various types, which can be differentiated by aggressive and indolent courses. During the past 15 years, great progress has been made in the treatment of B-NHL. Standard therapy is antibody / chemotherapy combinations, either alone or in combination with autologous stem cell transplantation, immunomodulatory drugs, radiation, proteasome inhibitors, signaling pathway inhibitors, and, in a very small number of patients, allogeneic stem cell transplantation. In many cases of B-NHL, the median age at diagnosis is greater than 55-60 years and the presence of comorbidities precludes intensive long-term chemotherapy or even allogeneic bone marrow transplantation.

[0007] The advent of adoptive CAR-T cell therapy targeting the CD19 antigen, widely expressed on the surface of lymphoma B cells, has made it possible to overcome these limitations, and currently there are about 20 studies of CD19 CAR-T cells registered with the FDA for the treatment of B-NHL and B-ALL. Although, to the inventors' knowledge, major breakthroughs were already achieved in 2011 in clinical trials for CLL and in 2013 in clinical trials for B-ALL, only recently have biomedical companies been allowed to use the same CD19 CAR products in Germany. In other countries in the EU (e.g. Austria), clinical trials with CD19 CAR T cells are being performed. More importantly, resistance has developed in anti-CD19 antibody or CAR-T cell therapy for B-NHL due to antigen loss. As treatment resistance has been observed after multiple chemotherapy / immunotherapy rounds, alternative targeting structures are urgently desired.

[0008] For the disease multiple myeloma, two anti-BCMA-CAR products have been previously reported and are in phase I clinical trials. These trials do not prove the applicability of anti-BCMA-CARs in B-NHL. For B-NHL, anti-BCMA targeted therapy is a possible alternative, especially when anti-CD19 CARs fail. Other immunotherapy strategies targeting multiple myeloma and undergoing clinical trials are anti-CD19 CARs, NY-ESO1, and TCR-transduced T cells directed towards MAGE-A1. Very different from BCMA as a tumor target, the number of patients available for selection is much smaller, since these target antigens are expressed in less than 10% of cases. Other targeted therapies include anti-CD38 and anti-SLAMF7 antibodies, but these therapies are conceptualized quite differently, since antibodies are not autonomous, do not form memory, and to our knowledge have not yet been proven to be involved in sufficient tumor eradication.

[0009] In addition, the ability to specifically target plasma cells would be of great benefit in the treatment of autoimmune diseases. Mild forms of autoimmune diseases are usually initially treated with nonsteroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). More severe forms of systemic lupus erythematosus (SLE), which involve organ dysfunction due to ongoing disease, are usually treated with steroids in combination with strong immunosuppressants, such as cyclophosphamide, a cytotoxic agent that targets circulating cells.

[0010] More recently, belimumab, an antibody targeting the cytokine BAFF (found at high levels in the serum of patients with autoimmune diseases), has been approved by the Food and Drug Administration (FDA) for use in SLE. However, only newly formed B cells depend on BAFF to survive in the human body, while memory B cells and plasma cells are less sensitive to selective BAFF inhibition (Jacobi et al. (2010) Arthritis Rheum 62:201-210). For rheumatoid arthritis (RA), TNF inhibitors were the first biologic agents approved, followed by abatacept, rituximab, tocilizumab, and others. These suppress key inflammatory pathways involved in joint inflammation and destruction, but at the cost of increased risk of infection due to relative immune suppression (Chan et al. 2010 Nat Rev Immunol 10:301-316; Keyser 2011 Curr Rheumatol Rev 7:77-87).

[0011] More recently, CAR-T cells have also been discussed as a targeted approach to treat autoantibody-mediated diseases (Ellebrecht et al. (2016) Science 353:179-184). Long-lived adherent plasma cells, which reside in a survival niche in the bone marrow, are often resistant to conventional immunosuppressive and cytotoxic drugs and to therapies targeting B cells and their activation. Rituximab in particular does not seem suitable for such treatment, since its target antigen, CD20, is not expressed on the surface of plasma cells. This therapeutic challenge could be met by using anti-BCMA CAR-T cell constructs, since BCMA is expressed on the surface of long-lived plasma cells.

[0012] To date, numerous other anti-BCMA CAR constructs have been reported in the field. In 2013, James N. Kochenderfer's group published the first anti-BCMA CAR-transduced T cell approach, a preclinical study using in vitro assays and mouse testing (Carpenter et al., 2013; Clin Cancer Res; vol. 19(8); pp. 2048-2060). In June 2015, Bluebird Bio and Celgene declared their focus on BCMA CAR-T cell therapy, which they are co-developing. Recruitment for a phase I clinical trial for multiple myeloma patients began in January 2016. In early 2016, the Abramson Cancer Center at the University of Pennsylvania began recruiting participants for a phase I trial using anti-BCMA CAR-transduced T cells to treat patients with multiple myeloma (ClinicalTrials.gov identifier: NCT02546167). CARs directed against BCMA are described in WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760. WO 2015 / 128653 also discloses CAR sequences that bind to BCMA. The part of the CAR involved in epitope recognition is a variant of the APRIL ligand, which shows improved binding to BCMA compared to wild type APRIL. An alternative therapeutic strategy involves anti-CD38 CARs. BCMA-binding antibodies are disclosed in WO 2015 / 166073 and WO 2014 / 068079.

[0013] Although a number of potential alternative therapeutic approaches are under development, there remains a great need to provide effective means to address medical disorders associated with the presence of pathogenic B cells, particularly multiple myeloma, non-Hodgkin's lymphoma, and autoantibody-dependent autoimmune diseases. Summary of the Invention

[0014] In light of the prior art, the technical problem underlying the present invention was to provide agents suitable for the treatment of diseases associated with pathogenic B cells.

[0015] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0016] The present invention thus relates to an isolated chimeric antigen receptor (CAR) polypeptide, the CAR comprising: an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to an iB cell maturation antigen (BCMA) polypeptide; ii. a transmembrane domain; iii. Intracellular domain and binds to an epitope comprising one or more amino acids between residues 13 and 32 of the N-terminus of human BCMA.

[0017] Thus, the present invention relates to genetically modified immune cells, preferably T cells, that express a CAR of the present invention and the use of such cells in the treatment of medical disorders associated with the presence of pathogenic B cells.

[0018] Thus, the present invention provides autologous T cells, preferably autologous T cells, suitable for transplantation, comprising anti-BCMA CARs for the treatment of different stages of mature B-NHL and multiple myeloma. In a preferred embodiment of the immunotherapy approach of the present invention, patient-derived T cells are transduced, preferably by retrovirus, to express the artificial immune receptors described herein, consisting of an antigen recognition moiety derived from an extracellular antibody fused to a transmembrane compartment followed by an intracellular signaling domain. The constructs described herein confer antitumor cytolytic capabilities to the transduced T cells.

[0019] As shown for other CAR-T cell delivery in the clinic, the present invention is characterized in that the CAR-based anti-BCMA CAR-T cells described herein have predictable, tolerable and manageable side effects. Preclinical studies of the anti-BCMA CAR-T cells described herein show selectivity for the tumor-associated antigen BCMA. T cells equipped with anti-BCMA CARs have high affinity and avidity, recognizing and destroying multiple myeloma cells while leaving normal hematopoietic cells unaffected. In a preferred embodiment, the transplantation of autologous T cells avoids the possibility of graft-versus-host disease. The formation of memory CAR-T cells, which is important in preventing relapse, may develop.

[0020] The anti-BCMA CAR-T cells described herein have high affinity and avidity, allowing them to recognize even mature B-NHL with low BCMA expression, thereby enabling T cell activation and tumor cell killing.

[0021] In preferred embodiments, such mature B-NHL includes some stages of FL (follicular lymphoma), DLBCL (diffuse large B-cell lymphoma), mantle cell lymphoma (MCL), and CLL (chronic lymphocytic leukemia).

[0022] The antigen recognition portion of the CAR described herein is preferably based on the humanized antibody described in WO / 2015 / 166073. The antibodies described therein were used to construct a number of CAR constructs that retain high affinity and specificity for BCMA. High affinity and specificity provide an advantage over other BCMA CAR constructs, as it allows for reduced off-target reactivity. It was a surprising result that the high specificity and affinity of the original antibody could be maintained in the CAR described herein, even targeting B cells with very low expression of the BCMA antigen.

[0023] The CAR of the invention preferably binds to an epitope comprising one or more amino acids at the N-terminus of BCMA, residues 13 to 32. In alternative embodiments, it may bind to other epitopes of BCMA, particularly the N-terminus of BCMA.

[0024] The present invention also includes various signaling domains. The exchange of signaling domains meets the requirements of a strong and rapid effector phase (CD28 costimulatory domain) or long-lasting relapse control guaranteed by T cell memory population (4-1BB signaling domain). As shown herein, the exchange of various signaling domains into many configurations allows CAR design flexibility without losing advantageous binding properties.

[0025] The anti-BCMA CAR-T cell products described herein are characterized by unique properties. Due to the low nanomolar affinity of the extracellular domain of the CAR-T cell construct, the anti-BCMA CARs described herein have an unparalleled high affinity, conferring extreme specificity and avidity to T cells. These properties allow the tumor cells to i) recognize, ii) activate against and iii) kill tumor cells with high surface expression of BCMA and surprisingly, tumor cells with low surface expression of BCMA.

[0026] The number of BCMA antigens expressed on the surface of tumor cells can be quantified using anti-BCMA antibodies coupled to a fluorescent dye in combination with Quantibrite beads (from Becton Dickinson). The preferred method used to quantitate the BCMA antigens expressed on the surface of tumor cells is "fluorescence-activated cell sorting / cell analysis" (FACS). The fluorescence intensity of the beads precisely correlates with the number of fluorescent antibodies bound to the cells, and is therefore an indication of the number of BCMA molecules on the cell surface. The fluorescence density associated with myeloma cells is typically at least 2-3 log higher than that of low-fluorescent B-NHL cells. 10 This is because the density of BCMA antigen is at least 2–3 log 10This indicates that the variation can be in the range of 2x.

[0027] It was found that none of the competing anti-BCMA CARs are reactive with B-NHL other than multiple myeloma cells or, in very rare cases, Burkitt's lymphoma. Thus, anti-BCMA CARs are reactive to an unprecedented variety of B-NHL. These properties are unexpected and surprising advantages for CAR-T therapy. According to typical expectations of those skilled in the art, it is necessary to express a large amount of target antigen in order to enable CAR-T targeting. CAR-T using the CAR of the present invention shows unprecedented activity against B cells with low expression levels of target antigen.

[0028] In a preferred embodiment, in combination with the MP71-vector and gamma-retroviral expression system, exceptionally high transduction rates of human T cells can be achieved.

[0029] Preferred embodiments regarding CAR-bound BCMA epitopes The CAR of the invention is preferably directed against an epitope comprising one or more amino acids at the N-terminus of human BCMA, residues 13 to 32. The amino acid sequence of residues 13 to 32 of CD269 is shown in SEQ ID NO: 33. The N-terminus of CD269 is shown in SEQ ID NO: 32. The extracellular domain of CD269 is presented in SEQ ID NO: 31.

[0030] To generate the binding specificity of the murine and chimeric antibodies described herein and previously (WO / 2014 / 068079) modified for use in the CAR format in the present invention, an antigen comprising the extracellular domain of CD269 as set forth in SEQ ID NO: 31 was used in vaccination. When generating antibodies, if the entire CD269 protein or a fragment thereof comprising the membrane-associated or intracellular domain is used as an antigen, antibodies can be generated that bind to the hidden or intracellular domain of CD269, making such drugs unsuitable or disadvantageous for therapeutic use. Thus, the CAR of the present invention is characterized by binding to the extracellular portion of CD269. The specific epitope within the extracellular domain is also a novel and unexpected preferred feature of the present invention.

[0031] Fab fragments prepared from murine or chimeric antibodies from which the CARs of the invention are derived were crystallized in complex with purified BCMA extracellular domain and the complex structure was dissolved. Structural analysis revealed detailed information about the epitope of the antibody / CAR binding region of the invention and its biological importance. The binding of the antibodies of the invention to an epitope comprising one or more amino acids of residues 13-32 of BCMA in the extracellular domain is an advantageous property due to its large binding specificity and extracellular location. To the inventors' knowledge, CARs binding to this region have not been reported before.

[0032] In one embodiment, the CAR of the invention is characterized in that it binds to an epitope comprising one or more of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 32 of CD269 (BCMA). In another embodiment, the CAR of the invention is characterized in that it binds to an epitope consisting of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 32 of CD269 (BCMA). These residues represent the amino acids that directly interact with the antibodies of the invention, as confirmed by the crystal structure data presented herein. The numbering of these residues is performed relative to SEQ ID NO: 32, which gives the sequence of the N-terminus of BCMA.

[0033] As already disclosed, the affinity of the antibody from which the CAR of the present invention is derived is surprisingly high and is superior to similar approaches attempted in the prior art. Thus, the CAR of the present invention is characterized by a high affinity not seen in other anti-BCMA CAR molecules. A Kd in the pM range (as described below) is generally accepted as a remarkable affinity that is not generally expected.

[0034] In another aspect, the humanized antibody or humanized antibody fragment from which the CAR of the present invention originates binds with high affinity to BCMA.When measured by surface plasmon resonance (e.g., Biacore), the antibody binds to human BCMA with an affinity of 100 nM, 90, 80, 70, 60, 50, 40, 30 nM or less, or 20 nM or less, or 15 nM or less, or 5 nM or less, or 1000 pM or less, or 500 pM or less, or 100 pM or less, or 80 pM or less, or for example, about 50 pM.Therefore, the CAR of the present invention shows the corresponding affinity.

[0035] In yet another embodiment, the antibody from which the CAR of the invention is derived binds to human CD269 with an affinity of about 1 pM to about 100 nM, or about 100 pM to about 50 nM, or about 200 pM to about 20 nM, as measured by surface plasmon resonance (e.g., Biacore), and thus the CAR of the invention exhibits the corresponding affinities.

[0036] In one embodiment, the CAR and / or CAR-T of the invention bind to cells expressing BCMA and express BCMA with a 1 / 1 log decrease compared to multiple myeloma cells, preferably compared to the multiple myeloma cell lines used in the examples presented herein. 10 ~1 / 4 log 10 , preferably 1 / 2 log 10 ~1 / 3 log 10The present invention is characterized in that it is capable of detecting BCMA present on the surface of cells in amounts of 0.01 to 0.01%. Non-limiting examples of such cells are non-Hodgkin's lymphoma (B-NHL) cells, such as the DOHH-2 cell line, and / or the SU-DHL4 cell line, and / or the JEKO-1 cell line, and / or the JVM-3 cell line, and / or the MEC-1 cell line.

[0037] Preferred embodiments regarding the CAR sequence: In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention comprises an extracellular antigen-binding domain comprising: - a heavy chain complementarity determining region 1 (H-CDR1) that has at least 80% sequence identity with SEQ ID NO: 1 (GFTFSRYW); - a heavy chain complementarity determining region 2 (H-CDR2) that has at least 80% sequence identity with SEQ ID NO: 2 (INPSSSTI); - a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity with SEQ ID NO: 3 (ASLYYDYGDAYDY); and a variable heavy chain (VH) comprising - a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity with SEQ ID NO: 4 (QSVESN); - a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity with SEQ ID NO:5 (SAS); - a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity with SEQ ID NO: 6 (QQYNNYPLT); The antibody is characterized in that it comprises a variable light chain (VL) comprising:

[0038] In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention comprises an extracellular antigen-binding domain comprising: - a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity with SEQ ID NO: 25 (RYWFS); - a heavy chain complementarity determining region 2 (H-CDR2) that has at least 80% sequence identity with SEQ ID NO: 26 (EINPSSSTINYAPSLKDK); - a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity with SEQ ID NO: 27 (SLYYDYGDAYDYW); and a variable heavy chain (VH) comprising - a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity with SEQ ID NO: 28 (KASQSVESNVA); - a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity with SEQ ID NO: 29 (SASLRFS); - a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity with SEQ ID NO: 30 (QQYNNYPLTFG); The antibody is characterized in that it comprises a variable light chain comprising:

[0039] The above CDR sequences shown as SEQ ID NOs: 25-30 represent embodiments obtained using alternative parameters for defining the CDR regions, and include, for example, additional flanking amino acids compared to SEQ ID NOs: 1-6.

[0040] The CDR sequences of SEQ ID NOs: 1-6 and 25-30 may also be determined such that polypeptide sequences that are at least 70%, 75%, 80%, 85%, 90%, or at least 95% identical to the specific sequences listed are included in the present invention.

[0041] In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention comprises the CDR sequence: - GFTFSRYW (H-CDR1; SEQ ID NO: 1), - INPX2X3STI (H-CDR2; SEQ ID NO: 7), where X2X3 is SS, NS, TS, GS, KS, RS, SD, SN, DE, - ASLYX4DYGDAX5DY (H-CDR3; SEQ ID NO: 8) (wherein X4 is Y, L, A, V, F, I, W and / or X5 is Y, L, F, I, V, A, C) and a VH domain comprising CDR sequences: - QSVX1X2N (L-CDR1; SEQ ID NO: 9) (wherein X1X2 is ES, SS, TS, QS, HS, DH), - SAS (L-CDR2; SEQ ID NO: 5), - QQYNNYPLTFG (L-CDR3; SEQ ID NO: 10) The antibody is characterized in that it comprises a VL domain comprising the following:

[0042] In an alternative embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention comprises the CDR sequence: - RYWX1S (H-CDR1; SEQ ID NO: 34) (wherein X1 is I, F, L, V, Y, C, G, A, S, T), - EINPX2X3STINYAPSLKDK (H-CDR2; SEQ ID NO: 35) (wherein X2X3 are SS, NS, TS, GS, KS, RS, SD, SN, DE), - SLYX4DYGDAX5DYW (H-CDR3; SEQ ID NO: 36) (wherein X4 is Y, L, A, V, F, I, W and / or X5 is Y, L, F, I, V, A, C) and a VH domain comprising CDR sequences: - KASQSVX1X2NVA (L-CDR1; SEQ ID NO: 37) (wherein X1X2 is ES, SS, TS, QS, HS, DH), - SASLRFS (L-CDR2; SEQ ID NO: 29), - QQYNNYPLTFG (L-CDR3; SEQ ID NO: 30) The antibody is characterized in that it comprises a VL domain comprising the following:

[0043] The above CDR sequences shown as SEQ ID NOs: 34-37 represent embodiments obtained using alternative parameters for defining the CDR regions, and include, for example, additional flanking amino acids compared to SEQ ID NOs: 1-6.

[0044] In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention has the following sequence: - H-CDR1: GFTFSRYW (SEQ ID NO: 1), - H-CDR2: INPSSSTI (SEQ ID NO: 2), - H-CDR3: ASLYYDYGDAYDY (SEQ ID NO: 3), - L-CDR1: QSVESN (SEQ ID NO: 4), - L-CDR2: SAS (SEQ ID NO: 5), - L-CDR3: QQYNNYPLT (SEQ ID NO: 6) The present invention is characterized by comprising:

[0045] In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention comprises the CDR sequence: - H-CDR1: RYWFS (SEQ ID NO: 25), - H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO: 26), - H-CDR3: SLYYDYGDAYDYW (SEQ ID NO: 27), - L-CDR1: KASQSVESNVA (SEQ ID NO: 28), - L-CDR2: SASLRFS (SEQ ID NO: 29), - L-CDR3: QQYNNYPLTFG (SEQ ID NO: 30) The present invention is characterized by comprising:

[0046] In one embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention has the sequence set forth in SEQ ID NO: 11 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINP SSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS) and a VH domain with at least 80% sequence identity; SEQ ID NO:12 (EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIY SASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK).

[0047] SEQ ID NOs: 11 and 12 represent the "full-length" VH and "full-length" VL domains of preferred CARs. Sequences with at least 70%, preferably 80%, 85%, 90%, or at least 95% sequence identity to SEQ ID NOs: 11 and 12 are included within the scope of the invention, particularly when such sequence variants exhibit the desired BCMA binding specificity (similar / equivalent function).

[0048] In one embodiment, an isolated chimeric antibody receptor (CAR) polypeptide comprising the VH and VL sequences of SEQ ID NOs: 11 and 12, or sequences at least 80% identical to SEQ ID NOs: 11 and 12, comprises at least W36, E50, L99, Y100, Y101, A106 of SEQ ID NO: 11 and at least S31, A34, S50, L53, Q89, Y91, Y94, L96 of SEQ ID NO: 12.

[0049] The amino acid residues listed above are those known to directly interact with the target BCMA epitope. Thus, the present invention relates to CARs in which sequence variations in VH and VL occur within a range of sequences that are at least 70%, preferably 80%, 85%, 90%, or at least 95% identical to SEQ ID NOs: 11 and 12, but at least the amino acid residues known to interact with the target epitope are included in the VH and VL domains.

[0050] In one embodiment, an isolated chimeric antibody receptor (CAR) polypeptide comprising the VH and VL sequences of SEQ ID NOs: 11 and 12, or sequences at least 80% identical to SEQ ID NOs: 11 and 12, comprises at least the CDR sequences of SEQ ID NOs: 1, 7, 8, 9, 5, 10, preferably the CDR sequences of SEQ ID NOs: 1-6, as described herein.

[0051] The present invention thus relates to CARs comprised in the VH and VL domains at least the CDR sequences described herein, although sequence variations in VH and VL occur to the extent that the sequences are at least 70%, preferably 80%, 85%, 90% or at least 95% identical to SEQ ID NOs: 11 and 12. CDRs refer to any of the sequences designated CDR herein, in particular SEQ ID NOs: 1-6 or SEQ ID NOs: 25-30.

[0052] In a preferred embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the present invention is characterized in that, when the CAR is expressed in a genetically modified immune cell, preferably a T lymphocyte, the immune cell is activated by binding to BCMA on the surface of non-Hodgkin's lymphoma (B-NHL) through the CAR, thereby inducing cytotoxic activity against the B-NHL.

[0053] In a preferred embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the invention is characterized in that the B-cell lymphoma is a non-Hodgkin's lymphoma (B-NHL) cell, such as the JeKo-1 cell line, and / or the DOHH-2 cell line, and / or the SU-DHL4 cell line, and / or the JVM-3 cell line, and / or the MEC-1 cell line.

[0054] The CAR of the present invention is characterized by the surprising property that even when the level of BCMA on the surface of a cell is very low, it can lead to CAR binding, T cell activation, and cell killing against the bound cell.This is a significant advantage compared to commonly reported CARs.Typically, CARs require a large number of surface antigens to enable cytotoxic activity following activation.Therefore, the CAR of the present invention is accompanied by an unexpected advantage in light of the CARs known in the prior art.

[0055] Derivatization of the above murine and / or chimeric and / or human antibodies to generate the CARs described herein can provide this advantage in some embodiments. In some embodiments, the characteristics of the BCMA epitope facilitate this advantage. In another embodiment, the high affinity and specificity of the VH and VL fragments described herein allows the sensitivity of the CARs of the invention. However, this property was unexpected to occur in combination with CARs from previously reported antibodies. It was quite surprising that the individual sequences presented herein, preferably the CDR regions of the VL and VH regions involved in binding, show sufficient specificity and strong binding to activate and direct CAR-T cells to cells with very low expression of BCMA.

[0056] It was unexpected that the VH and VL fragments described herein can be arranged in a variety of configurations in the CARs described herein while still maintaining high specificity and high affinity for the target epitope. As shown below and in Figure 3, the CARs can be arranged in a VH-VL or VL-VH configuration and have variations in the linker, and / or hinge, and / or transmembrane domain, and / or costimulatory domain, and / or activation domain while still maintaining efficacy. This surprising feature of the invention allows for more flexibility in designing CARs directed to BCMA, allowing for further modification and / or optimization of the CAR structure based on the VH and VL domains described herein, should further developments become necessary or desirable.

[0057] In a preferred embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the present invention is characterized in that when expressed in a genetically modified immune cell (preferably a T-lymphocyte), the immune cell binds to BCMA on the surface of multiple myeloma (MM) cells through the CAR and is activated, thereby inducing cytotoxic activity against the MM cells. Preferred MM cells are those disclosed herein. In a preferred embodiment, the CAR described herein exhibits effective binding and cytotoxic activity against both MM and B-NHL. In light of the prior art, it would not have been expected by one skilled in the art that the CAR described herein could exhibit activity against both types of cells.

[0058] Preferred embodiments regarding humanized VH and VL domains As disclosed in detail herein and previously (WO / 2015 / 166073), the sequence of antibody J22.9-xi was humanized to provide a reagent more suitable for administration to human subjects. Various humanized sequence variants of J22.9-xi were generated and tested for their binding affinity and specificity to both human BCMA and cynomolgus BCMA. In a preferred embodiment, the CAR of the present invention incorporates these humanized sequences. The results of binding assays performed with the corresponding antibodies reveal that the humanized sequences maintain the desirable binding properties of the chimeric reagent J22.9-xi. In the sequences below, the underlined regions represent CDRs or pseudo-CDRs depending on which method was used to determine the CDRs.

[0059] Preferred embodiments regarding humanized VH variants Additional information regarding the humanized VH and VL sequences that are preferably incorporated by the CAR of the present invention is provided below.

[0060] Chimeric sequence: HC Mouse (SEQ ID NO: 38): QVQLQQSGGGLVQPGGSLKLSCAASGIDFS RYWMS WVRRAPGKGLEWIG EINPDSSTINYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCA SLYYDYGDAMDYW GQGTSVTVSS

[0061] The HC mouse sequence is a heavy chain (VH) variable region originally developed for the chimeric antibody J22.9-xi, which contains a VL domain and a VH domain obtained from a mouse antibody and can bind to an epitope in the extracellular domain of CD269 (BCMA), and the VL domain and the VH domain are fused to human CL domain and CH domain, respectively. In some embodiments, a CAR can comprise this HC mouse sequence or its CDRs.

[0062] Partially humanized sequences: Partially humanized HC (SEQ ID NO: 39): EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYWMS WVRQAPGKGLEWVG EINPDSSTINYAPSLKGR FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0063] This partially humanized HC sequence represents an altered amino acid sequence (through amino acid substitutions) compared to the chimeric antibodies disclosed herein, whereby the VL and VH binding regions have been altered relative to their sequences to make them more suitable for administration to humans.

[0064] Humanized VH sequence: hHC01 (SEQ ID NO: 40) EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWMS WVRQAPGKGLVWVG EINPDSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0065] Humanized VH sequence with removed post-translational modification motifs: hHC02 (SEQ ID NO:41) EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWX 1 S WVRQAPGKGLVWVG EINPX 2 X 3 STINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYX 4 DYGDAX 5 D.Y.W. GQGTLVTVSS however, X1: I, F, L, V, Y, C, G, A, S, T, preferably I or F; and / or X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS; and / or X4: Y, L, A, V, F, I, W, preferably Y; and / or X5: Y, L, F, I, V, A, C, preferably Y.

[0066] The humanized sequences "hHC01" and "hHC02" represent preferred amino acid sequences for the CAR of the present invention and have altered sequences compared to both the original chimeric sequence and the partially humanized sequence described herein.

[0067] The PTM mutations are intended to remove potentially harmful post-translational modification motifs from the protein while maintaining favorable binding properties. Preferably, positions 1, and / or 5, and / or 6, and / or 19, and / or 27, and / or 28, and / or 34, and / or 39, and / or 46, and / or 48, and / or 54, and / or 69, and / or 84, and / or 85, and / or 86, and / or 88, and / or 93, and / or 107, and / or 115 of hHC01 and hHC02 are mutated (substituted) compared to the original chimeric sequence. The significance of the substitutions mainly concerns the resulting amino acid, and not the original amino acid. Thus, the changes may also occur from the corresponding amino acid of the original chimeric sequence or other variants (e.g. partially humanized sequences).

[0068] The following substitutions are preferred in some embodiments and differ compared to the chimeric sequence (SEQ ID NO:38): - the amino acid M34 of the HC (VH) sequence is replaced by any amino acid, preferably I, L, F, V, Y, C, G, A, S, T; and / or - the amino acid E46 of the HC (VH) sequence is substituted by V; and / or - amino acids D54 and S55 of the HC (VH) sequence are replaced by any combination of amino acids, preferably SS, TS, GS, KS, RS, SD, SN, DE; and / or - the amino acid Y101 of the HC (VH) sequence is substituted with any amino acid, preferably L, A, V, F, I, W; and / or the amino acid M107 of the HC (VH) sequence is substituted with any amino acid, preferably L, Y, F, I, V, A, C.

[0069] Sequences that may have altered positions of residues required for direct interaction with BCMA: hHC03 - changed amino acids involved in interaction with BCMA (SEQ ID NO:42): NYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SX 5 X 6 X 7 DYGDX 8 M.D.Y.W. GQGTLVTVSS However, the preferred amino acids are X1: W, F, Y, preferably W; and / or X2: S, T, N, Q, D, E, preferably S; and / or X3: W, F, Y, preferably W; and / or X4: E, Q, preferably E; and / or X5: L, I, V, G, A, preferably L; and / or X6: Y, X, preferably Y; and / or X7: Y, F, L, I, V, M, preferably Y; and / or X8: A, G, V, preferably A.

[0070] The "hHC03" humanized sequence represents a preferred amino acid sequence that includes amino acid sequences that are altered compared to both the original chimeric and partially humanized sequences. These changes in sequence are intended to reflect potential changes in replaceable amino acids that bind to the BCMA target while maintaining favorable binding properties. The significance of the substitutions relates primarily to the resulting amino acid, not the original amino acid. Thus, changes may occur from the corresponding amino acid in the original chimeric sequence or other variants.

[0071] for example: - amino acid W33 of the HC(VH) sequence is W, F, Y; and / or - amino acid S35 of the HC (VH) sequence is S, T, N, Q, D, E; and / or - amino acid W47 of the HC(VH) sequence is W, F, Y; and / or - amino acid E50 of the HC (VH) sequence is E, Q; and / or - amino acid L99 of the HC (VH) sequence is L, I, V, G, A; and / or - amino acid Y100 of the HC(VH) sequence is Y,X; and / or - the amino acid Y101 of the HC (VH) sequence is Y, F, L, I, V, M; and / or - Amino acid A106 of the HC (VH) sequence is A,G,V.

[0072] In general, any changes in the CDR regions that occur during humanization can also be considered as a feature of the CDR sequences when considered independently of the overall framework sequences. Such modified CDR sequences can be considered as defining features of the invention in the context of the overall framework regions described herein or independently of that context. For example, the underlined CDR sequences in hHC01-hHC03 can be considered as defining features of the invention independently of the surrounding variable region sequences.

[0073] Examples of humanized HC (VH) sequences: hHC04 (SEQ ID NO:43): EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS hHC05 (SEQ ID NO:44): EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWFS WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS hHC06 (SEQ ID NO:45): EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS hHC07 (SEQ ID NO:46): EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWFS WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0074] To eliminate a potential post-translational modification site in humanized J22.9, residue D54 in the heavy chain CDR2 was mutated to asparagine (N) to create a new potential modification site for N-linked glycosylation (e.g., hHC04, hHC05). The heavy chain containing the mutated N54 can be glycosylated. However, the corresponding IgG, J22.9-FNY, bound to BCMA in FACS and ELISA, and was crystallized in complex with BCMA. It was surprising that such a large extension of the side chain did not prevent binding to BCMA, and this observation led us to predict that many different amino acid substitutions would be tolerated at this position, potentially including derivatizations other than sugars.

[0075] alignment: A CLUSTAL W (1.83) multiple sequence alignment of various substitution positions within the HC sequence provides the appropriate sequence comparison shown in Figure 13. A "generic sequence" represents one HC sequence in which each X represents a potential amino acid change for any given amino acid. Preferred amino acid substitutions are those described above at each of the potential mutation positions.

[0076] Preferred embodiments regarding humanized VL variants Chimeric sequence: LC mouse (SEQ ID NO: 47): DIVMTQSQRFMTTSVGDRVSVTC KASQSVDSNVA WYQQKPRQSPKALIF SASLRFS GVPARFTGSGSGTDFTLTISNLQSEDLAEYFC QQYNNYPLTFG AGTKLELKR

[0077] The LC mouse sequence represents the variable region of the light chain (VL) originally developed for the chimeric antibody J22.9-xi, and contains the VL and VH domains obtained from a mouse antibody and can bind to an epitope in the extracellular domain of CD269 (BCMA). In some embodiments, the mouse VL domain or its CDRs can be used in the CAR of the invention.

[0078] Partially humanized sequences: Partially humanized LC (SEQ ID NO: 48): DIVMTQSPATLSVSVGDEVTLTC KASQSVDSNVA WYQQKPGQAPKLLIY SDDLRFS GVPARFSGSGSGTDFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0079] Partially humanized LC sequences represent sequences that have been altered (through amino acid substitutions) compared to the chimeric antibody, whereby the VL and VH binding regions have been altered relative to their sequences to render them in a form more suitable for administration to humans.

[0080] Humanized VL sequence: hLC01 (SEQ ID NO:49): EIVMTQSPATLSVSPGERATLSC KASQSVDSNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR Humanized VL sequences with removed post-translational modification motifs: hLC02 (SEQ ID NO:50): EIVMTQSPATLSVSPGERATLSC KASQSVX 1 X 2 NVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR however, X1X2: ES, SS, TS, QS, HS, DH, preferably ES.

[0081] The humanized sequences "hLC01" and "hLC02" represent preferred amino acid sequences that contain altered amino acid sequences compared to both the original chimeric and partially humanized sequences described herein.

[0082] The PTM mutations are intended to remove potentially deleterious post-translational modification motifs from the protein while maintaining favorable binding properties. Preferably, positions 1, and / or 8, and / or 9, and / or 10, and / or 13, and / or 15, and / or 17, and / or 19, and / or 20, and / or 21, and / or 22, and / or 30, and / or 41, and / or 43, and / or 45, and / or 49, and / or 58, and / or 63, and / or 70, and / or 77, and / or 83, and / or 85, and / or 87 of hHC01 and hHC02 are mutated (substituted) compared to the original chimeric sequence. The significance of the substitutions mainly concerns the resulting amino acid, and not the original amino acid. Thus, the changes may also occur from the corresponding amino acid of the original chimeric sequence or other variants (e.g. partially humanized sequences).

[0083] The following substitutions are preferred and differ from the chimeric and partially humanized sequences: - the amino acid D1 of the LC(VL) sequence is substituted with E; and / or - the amino acid V15 of the LC (VL) sequence is substituted with P; and / or - amino acid D17 of the LC(VL) sequence is substituted with E; and / or - the amino acid V19 of the LC (VL) sequence is substituted with A; and / or - the amino acid T22 of the LC(VL) sequence is substituted with S; and / or - the amino acids D30 and S31 of the LC (VL) sequence are replaced by any combination of amino acids, preferably ES, SS, TS, QS, HS, DH; and / or - the amino acid V58 of the LC (VL) sequence is substituted with I; and / or - the amino acid D70 of the LC(VL) sequence is substituted by E.

[0084] Sequences that potentially alter the positions of residues required for direct interaction with BCMA within the CDR binding region: hLC03 - Changed amino acids involved in interaction with BCMA (SEQ ID NO:51): EIVMTQSPATLSVSPGERATLSC KASQSVDX 1 X 2 VX 3WX4QQKPGQAPRALIX5 X 6 AX 7 X 8 RX 9 S GIPARFSGSX 10 X 11 GTEFTLTISSLQSEDFAVYYC X 12 QX 13 NNX 14 PX 15 TFG AGTKLELKR However, the preferred amino acids are X1: S, H, T, N, D, Q; and / or X2: N, E, Q; and / or X3: A, G, V, S, T, L, I; and / or X4: Y, F, L, I, V, A, G; and / or X5: Y, F, L; and / or X6: S, T; and / or X7: S, T, D, N, H, E, Q; and / or X8: L, V, I, M; and / or X9: F, L, I, V, Y, M; and / or X 10 : G, X; and / or X 11 : S, X; and / or X 12 : Q, V, L, I, M; and / or X 13 : Y, F, L, I, Q; and / or X 14 : Y, F, R, Q, K; and / or X 15 :L, I, V, F.

[0085] The "hLC03" humanized sequence represents a preferred amino acid sequence that includes amino acid sequences that are altered compared to both the original chimeric sequence and the partially humanized sequence. These changes in sequence are intended to reflect potential changes in replaceable amino acids that bind to the BCMA target while maintaining favorable binding properties. The significance of the substitutions relates primarily to the resulting amino acid, and not to the original amino acid. Thus, changes may occur from the corresponding amino acid in the original chimeric sequence or other variants.

[0086] for example: - amino acid S31 of the LC(VL) sequence is S, H, T, N, D, Q; and / or - amino acid N32 of the LC(VL) sequence is N, E, Q; and / or - amino acid A34 of the LC(VL) sequence is A, G, V, S, T, L, I; and / or - amino acid Y36 of the LC(VL) sequence is Y, F, L, I, V, A, G; and / or - amino acid Y49 of the LC(VL) sequence is Y,F,L; and / or - amino acid S50 of the LC(VL) sequence is S,T; and / or - amino acid S52 of the LC(VL) sequence is S, T, D, N, H, E, Q; and / or - amino acid L53 of the LC (VL) sequence is L, V, I, M; and / or - amino acid F55 of the LC(VL) sequence is F,L,I,V,Y,M; and / or - amino acid G66 of the LC(VL) sequence is G,X; and / or - amino acid S67 of the LC(VL) sequence is S,X; and / or - amino acid Q89 of the LC(VL) sequence is Q,V,L,I,M; and / or - amino acid Y91 of the LC(VL) sequence is Y,F,L,I,Q; and / or - amino acid Y94 of the LC(VL) sequence is Y, F, R, Q, K; and / or - amino acid L96 of the LC(VL) sequence is L,I,V,F.

[0087] In general, any changes in the CDR regions that occur during humanization can also be considered as a feature of the CDR sequences when considered independently of the overall framework sequences. Such modified CDR sequences can be considered as defining features of the invention in the context of the overall framework regions described herein or independently of that context. For example, the underlined CDR sequences in hLC01-hLC03 can be considered as defining features of the invention independently of the surrounding variable region sequences.

[0088] Example of a humanized LC sequence: hLC04 (SEQ ID NO:52): EIVMTQSPATLSVSPGERATLSC KASQSVESNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0089] alignment: A CLUSTAL W (1.83) multiple sequence alignment of various substitution positions within the HC sequence provides the appropriate sequence comparison shown in Figure 14. A "generic sequence" represents one HC sequence in which each X represents a potential amino acid change for any given amino acid. Preferred amino acid substitutions are those described above at each of the potential mutation positions.

[0090] Thus, the present invention relates to the sequences of hHC01, and / or hHC02, and / or hHC03, and / or hHC04, and / or hHC05, and / or hHC06, and / or hHC07, and / or hLC01, and / or hLC02, and / or hLC03, and / or hLC04, or any combination thereof.

[0091] All possible combinations of the potential modifications presented herein with respect to any potential alternative residues (indicated by X in the "generic" sequences) are encompassed by the present invention. By combining one or more of these various substitutions, humanized variants exhibiting the desired binding characteristics of the originally developed chimeric antibodies presented herein can be generated. The antibodies or portions thereof described herein also include sequences that are at least 80%, preferably 90%, identical in sequence to the humanized sequences explicitly disclosed or disclosed through sequence formulas.

[0092] The present invention further comprises: X1VQLX2X3SGGGLVQPGGSLX4LSCAASGX5X6FX7X8YWZ1SWVRX9AP GKGLEWX 10 GEINPZ2SSTINYAPSLKX 11 X 12 FX 13 ISRDNAKNTLYLQMX 14 X 15 X 16 RX 17 EDTAX 18 YYCASLYYDYGDAZ3DYWGQGTX 19 X4: K, R; X5: I, F; X6: D, T; X7: S, D; X8: R, D; X9: R, Q; X10: Q, E; X11: Q, E; X12: Q, V; X13: Q, E; X14: K, R; X15: I, F; X16: D, T; X17: S, D; X18: R, D; X19: R, Q; X20: Q, V; X21: Q, E; X22: Q, V; X23: Q, E; X24: K, R; X25: I, F; X26: D, T; X27: S, D; X28: R, D; X29: R, Q; X30: Q, E; X31: Q, E; X32: Q, E; X33: Q, E; X34: K, R; X35: I, F; X36: Q 10 :I,V;X 11 :D,G;X 12 :K,R;X 13 :I,T;X 14 :S,N;X 15 :K,S;X 16 :V, L;X 17 :S,A;X 18 :L,V;X 19: S, L; at least one of Z1 is I, F, L, V, Y. C, G, A, S, T, preferably I or F; and / or Z2: S, N, T, G, K, R, D, preferably S; and / or Z3: Y, L, F, I, V, A, C, preferably Y).

[0093] This embodiment includes various humanized sequences for the CAR of the invention, in particular its VH sequence and any variant characterized by the advantageous humanization carried out in the CDRs described herein.

[0094] The present invention further comprises: DIVMTQSX1X2X3X4X5X6SVGDX7VX8X9TCKASQSVESNVAWYQQKPX 10 QX 11 PKX 12 LIX 13 SX 14 X 15 LRFSGVPARFX 16 GSGSGTDFTLTISX 17 LQSEDX 18 AX 19 YX 20 X4: M, L; X5: T, S; X6: T, V; X7: R, E; X8: S, T; X9: V, L; X10: V, P; X11: V, P; X12: V, P; X13: V, P; X14: V, P; X15: V, P; X16: V, P; X17: V, P; X18: V, P; X19: V, L; X20: V, P; X21: V, P; X22: V, P; X23: V, P; X24: V, P; X25: V, P; X26: V, P; X27: V, P; X28: V, P; X29: V, P; X30: V, P; X31: V, P; X32: V, P; X33: V, P; X34: V, P; X35: V, P; X36: V 10 :R,G;X 11 :S,A;X 12 :A,L;X 13 :F,Y;X 14 :A,D;X 15 :S,D;X 16 :T,S;X 17 :N,S;X 18 :L, F;X 19 :E,V;X 20 :F,Y).

[0095] This embodiment includes various humanized sequences for the CAR of the invention, in particular its VL sequence and any variant characterized by the advantageous humanization carried out in the CDRs described herein.

[0096] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention is characterized in that the extracellular binding domain comprises a linker polypeptide located between the VH domain and the VL domain, and the linker is preferably selected from a Whitlow linker (SEQ ID NO: 13; GSTSGSGKPGSGEGSTKG), a Gly-Ser linker (SEQ ID NO: 14; SSGGGGSGGGGSGGGGS), a linker having at least 80% sequence identity with SEQ ID NO: 13 or 14.

[0097] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention is characterized in that the CAR comprises a spacer polypeptide located between the extracellular binding domain and the transmembrane domain, and the selection of the spacer is preferably selected from: - IgG1-CD28 spacer (SEQ ID NO: 15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPK DTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK), - IgG1Δ - 4-1BB spacer (SEQ ID NO: 16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFP PKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK), - IgG4 (Hi-CH2-CH3) spacer (SEQ ID NO: 17; ESKYGPPCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), - IgG4 (Hi-CH3) spacer (SEQ ID NO: 18; ESKYGPPCPPCPGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), - IgG4 (Hi) spacer (SEQ ID NO: 19; ESKYGPPCPPCP), - a spacer having a sequence that is at least 80% identical to any one of SEQ ID NOs: 15 to 19 The present invention is characterized in that it is carried out from the following:

[0098] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention is characterized in that the selection of the transmembrane domain is preferably the CD8α domain (SEQ ID NO: 20; IYIWAPLAGTCGVL It is characterized in that it is composed of a CD28 domain (SEQ ID NO: 21; FWVLVVVGGVLACYSLLVTVAFIIFWV), a transmembrane domain having at least 80% sequence identity with SEQ ID NO: 20 or 21.

[0099] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention comprises an intracellular domain that comprises a costimulatory domain, and the choice of the costimulatory domain is preferably a 4-1BB costimulatory domain (SEQ ID NO: 22; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CEL), CD28 costimulatory domain (SEQ ID NO: 23; RSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRS), characterized in that it is made up of a costimulatory domain having a sequence that is at least 80% identical to SEQ ID NO: 22 or 23.

[0100] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention comprises a signaling domain, which signaling domain is preferably a CD3ζ (CD28 or 4-1BB) signaling domain (SEQ ID NO: 24; LRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or a signaling domain having at least 80% sequence identity with SEQ ID NO: 24.

[0101] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention is characterized in that the CAR comprises a tandem costimulatory domain comprising a 4-1BB costimulatory domain (SEQ ID NO: 22), a CD28 costimulatory domain (SEQ ID NO: 23), and a CD3 zeta signaling / activation domain (SEQ ID NO: 24).

[0102] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the invention is characterized in that the CAR comprises a leader sequence, the leader sequence being preferably selected from the IgK leader (SEQ ID NO: 55; MDFQVQIFSFLLISASVIMSR), the GMCSF leader (SEQ ID NO: 56; MLLLVTSLLLCELPHPAFLLI), a leader sequence that has at least 80% sequence identity with SEQ ID NO: 55 or 56.

[0103] Yet another aspect of the present invention is a)- a nucleotide sequence encoding an isolated chimeric antigen receptor (CAR) polypeptide as described herein, and / or - a nucleotide sequence comprising the sequences set forth in SEQ ID NO: 66 and / or 67 or SEQ ID NO: 86 to 94 a nucleic acid molecule comprising: b) a nucleic acid molecule complementary to the nucleotide sequence of a); c) a nucleic acid molecule comprising a nucleotide sequence that is sufficiently identical in sequence to be functionally similar / equivalent to the nucleotide sequence set forth in a) or b), preferably at least 80% identical to the nucleotide sequence set forth in a) or b); d) a nucleic acid molecule which, as a consequence of the degeneracy of the genetic code, results in the nucleotide sequence according to a) to c); e) A nucleic acid molecule according to a) to d) which has been altered by deletions, and / or additions, and / or substitutions, and / or rearrangements, and / or inversions, and / or insertions, but which is functionally similar / equivalent to the nucleotide sequences according to a) to d). The present invention relates to an isolated nucleic acid molecule selected from the group consisting of:

[0104] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0105] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0106] Yet another aspect of the present invention relates to a vector comprising a nucleic acid molecule as described herein, preferably a viral vector, more preferably a gamma retroviral vector.

[0107] Yet another aspect of the present invention relates to a genetically modified immune cell comprising a nucleic acid molecule or vector as described herein and / or expressing a CAR as described herein, preferably selected from the group consisting of T lymphocytes and NK cells, more preferably cytotoxic T lymphocytes.

[0108] In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecules or vectors described herein and / or expressing the CAR described herein are CD4 + and / or CD8 + T cells (CD4 + T cells and CD8 + These populations of T cells are preferably a mixture of CD4 + Transduced cells and CD8 + Compositions containing both transformed cells exhibit particularly effective cytolytic activity against a variety of malignant B cells (e.g., multiple myeloma and B-NHL), preferably against these cells and / or the associated medical disorders described herein.

[0109] In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecules or vectors described herein and / or expressing the CAR described herein are CD4 + T cells and CD8 + Preferably, the T cells are in a ratio of 1:10 to 10:1, more preferably 5:1 to 1:5, or 2:1 to 1:2, or 1:1. Modified CAR-T cells expressing CAR and directed against BCMA are injected at the above ratios, preferably CD4 + / CD8 + Dosing at a ratio of 1:1 leads to advantageous properties while treating the diseases mentioned herein, for example these ratios provide improved therapeutic response and reduced toxicity.

[0110] In a preferred embodiment, immune cells for administration in the treatment of the diseases mentioned herein are genetically modified with the nucleic acid molecules described herein that encode and express the anti-BCMA CARs described herein, using the "Sleeping Beauty" transposon system (particularly the Sleeping Beauty transposase). The Sleeping Beauty transposon system, in the context of the present invention, which aims to modify immune cells to express the CARs described herein, is a synthetic DNA transposon designed to introduce a precisely defined DNA sequence into a vertebrate chromosome. The Sleeping Beauty transposon combines the advantages of viruses with those of naked DNA. Viruses have been selected in evolution based on their ability to infect and replicate in new host cells. At the same time, cells have evolved major molecular defense mechanisms to protect themselves from viral infection. Avoiding the use of viruses is also important for social and regulatory reasons. Thus, the use of non-viral vectors such as the Sleeping Beauty system avoids many, if not all, of the defenses that cells use against vectors. For these reasons, the Sleeping Beauty system allows for a particularly effective and safe genetic modification of immune cells for administration to patients.

[0111] Yet another aspect of the present invention relates to an immune cell as described herein, comprising a nucleic acid molecule or vector as described herein and / or expressing a CAR as described herein, for use as a medicament in the treatment of a medical disorder associated with the presence of pathogenic B cells (e.g., a disease of plasma cells, and / or memory B cells, and / or mature B cells, in particular multiple myeloma and non-Hodgkin's lymphoma).

[0112] In one embodiment, the medical use of immune cells is characterized in that the medical disorder being treated is multiple myeloma.

[0113] In one embodiment, the medical use of immune cells is characterized in that the medical disorder being treated is non-Hodgkin's lymphoma.

[0114] In one embodiment, the medical use of immune cells is characterized in that the medical disorder to be treated is associated with pathogenic mature B cells. To the inventor's knowledge, no previous disclosures in the field are apparent that teach that such mature B cells can be an effective target of the BCMA CAR-T described herein. Some of the tested tumor cell lines shown in the examples below are associated with mature B cells, but not necessarily of the memory type. In contrast, immature B cells are considered to be the B cells that cause acute lymphoblastic leukemia. Thus, the present invention also includes a method of treating the medical disorder disclosed herein, which includes administering a therapeutically effective amount of a CAR or a therapeutic agent comprising the CAR of the present invention to a subject in need of such treatment.

[0115] Yet another aspect of the invention pertains to pharmaceutical compositions comprising a CAR, or a therapeutic agent comprising a CAR as described herein, in association with a pharma- ceutical acceptable carrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] Multiple myeloma (also called plasmacytoma) is a currently incurable B-cell lymphoma derived from a malignantly transformed plasma cell clone. The disease is the most frequent tumor of bone and bone marrow, with a median life expectancy of 7 years and accounting for 2% of annual cancer deaths. Malignant transformation is thought to occur at a developmental stage when B cells have completed VDJ rearrangement and isotype switching in the germinal centers of secondary lymphoid organs. The median age of diagnosis is 70 years. This indicates that many patients have comorbidities that preclude intensive and prolonged chemotherapy and radiotherapy. Furthermore, allogeneic bone marrow transplantation is usually excluded in this patient cohort. The disease is clinically characterized by osteolytic damage, hypercalcemia, hematopoietic failure, amyloid deposition, renal failure, excessive production of heavy and / or light chains, hyperviscosity, infections, and bleeding disorders. Standard treatment is chemotherapy alone or in combination with autologous stem cell transplantation, immunomodulatory agents (e.g., immunomodulatory drugs (IMIDs)), localized radiation, proteasome inhibitors, and, in a minority of patients, allogeneic stem cell transplantation. Despite the above modalities of intensive treatment, the disease usually recurs, with primary and secondary resistance occurring after multiple lines of therapy.

[0117] The adoptive chimeric antigen receptor (CAR)-T cell therapy described herein targeting B cell maturation antigen (BCMA) can overcome these limitations in multiple myeloma because BCMA is highly expressed on multiple myeloma tumor cells but not on normal B cells or precursor B cells. Secondly, resistance occurs in anti-CD19 antibody therapy or anti-CD19 CAR-T cell therapy for B cell non-Hodgkin's lymphoma (B-NHL) due to antigen loss. In B-NHL, treatment resistance occurs after multiple chemotherapy / immunotherapy, so alternative target structures are warranted. In mature B-NHL, BCMA is a suitable target, so anti-BCMA CAR-T with high affinity can be used for treatment, even in B-NHL, as specifically described below.

[0118] The transplantation of BCMA CAR-T cells against the tumor-associated antigen BCMA is selected and is applicable and effective in elderly patients and even after the emergence of multidrug resistance. The side effects of this transplantation are predictable, tolerable, and manageable. Autologous T cells with anti-BCMA CAR have high affinity and avidity, recognizing and destroying multiple myeloid cells while avoiding normal hematopoietic cells (e.g., T cells, B cells, and their myeloid precursors), as well as all myeloid cells and NK cells. Because it is an autologous transplant of T cells, graft-versus-host disease cannot occur. Anti-BCMA CAR-T cells can recognize even mature B-cell NHLs that have low expression of BCMA due to their high affinity and avidity, allowing T cell activation and tumor cell killing. Such mature B-NHLs include several stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

[0119] In some embodiments, the anti-BCMA CAR-T cells described herein can be applied to multiple myeloma and B-NHL patients who are not suitable for other therapies, more specifically, i) patients with multi-drug resistance, and / or ii) patients who are not suitable for allogeneic stem cell transplantation, and / or iii) patients with existing comorbidities that preclude further chemotherapy, and / or iv) elderly patients who cannot tolerate chemotherapy, and / or v) the CAR can be applied as a salvage therapy even after the disease has progressed and multiple other standard therapies have failed, and / or vi) the CAR can be applied even when the antigen density on the surface of the target tumor cells is so low that antibodies may not work, and / or vii) the structure of the original antibody in complex with BCMA at near atomic resolution demonstrates the incredible specificity of the CAR, which is biologically safe, a feature not seen with other anti-BCMA CAR-T cells, and / or viii) it can be applied as a monotherapy, which is not the case with antibodies.

[0120] For other anti-BCMA CAR-T cells described in the prior art, reactivity has only been seen in multiple myeloid cells and patients. Conversely, our anti-BCMA CAR has an unexpectedly large sensitivity even to B-NHL cell lines with low expression of BCMA. Our anti-BCMA CAR confers the extremely large avidity required for antitumor efficacy to T cells. No other anti-BCMA CAR has been reported to react with mature B-NHL, diffuse large B-cell lymphoma (DLBCL), certain stages of follicular lymphoma, mantle cell lymphoma, or chronic lymphocytic leukemia. In this invention, we demonstrate that our anti-BCMA CAR does not confer T cell reactivity against physiological B cells, T cells, NK cells, endothelial cells, or any myeloma cell line and its precursors. Thus, the present invention has unprecedentedly small off-target reactivity on the surface of other hematopoietic tissues. Unlike anti-CD38 CAR-T cells, our anti-BCMA CAR has no unwanted reactivity against myeloma cell precursors.

[0121] The amino acid sequence of the scFV fragment previously described in WO / 2015 / 166073 and WO / 2014 / 068079 was modified to i) allow folding and expression in the context of a transmembrane receptor structure, ii) reverse the order of the light and heavy chain fragments, and iii) lengthen the linker sequence between the heavy and light chains, allowing full expression at the surface of T cells while still maintaining proper binding of the antigen.

[0122] Due to the low nanomolar affinity of the original FSY IgG (antibody template for the scFv portion of the CAR-T cell construct), the present invention is characterized in a preferred embodiment by an anti-BCMA CAR with an unexpectedly high affinity, providing extremely high specificity and avidity for T cells. The high affinity and avidity allows the CAR-T cells to i) recognize, ii) be activated against, and iii) kill tumor target cells with high, medium and low surface expression of BCMA. None of the above-mentioned anti-BCMA CARs of the prior art have demonstrated reactivity against B-NHL other than multiple myeloid cells. Thus, the anti-BCMA CAR of the present invention is a specific and highly active reagent against an unprecedented variety of B-NHL with low / low levels of BCMA molecules.

[0123] In combination with retroviral vectors (preferably MP71-vectors) and gamma-retroviral expression systems, exceptionally high transduction rates can be achieved in human T cells.

[0124] Another distinct advantage of the present invention is the detailed knowledge of the BCMA epitope recognized by the scFv of the CAR. To date, no other antibody-based inventions or publications have identified the BCMA epitope. For example, the anti-BCMA CARs described herein exhibit a substantially greater biological safety profile and no known off-target reactivity in vivo or in vitro.

[0125] In addition, the inventors have swapped signaling elements of the CAR construct of the present invention in a simple three-step cloning process, allowing for modular composition of clinically applicable anti-BCMA CARs.

[0126] In an in vitro co-culture system, the anti-BCMA CAR-T cells of the invention become activated upon exposure to BCMA-expressing human B-NHL and multiple myeloma tumor cell lines, and these T cells develop an effector phenotype that secretes high levels of IFN-γ, a phenotype predictive of cytotoxic activity.

[0127] Preclinical evaluation will include i) in vitro cytotoxicity testing against appropriate B-NHL cell lines and primary myeloma cells from patients and ii) in vivo testing of anti-BCMA CAR activity against xenografted B-NHL and multiple myeloma cell lines.

[0128] In an in vivo human setting, myeloma patients with the following characteristics will be evaluated through a Phase 1 clinical trial: i) multi-drug resistant patients, ii) patients unsuitable for allogeneic stem cell transplant, iii) patients with existing comorbidities precluding further chemotherapy, iv) elderly patients unable to tolerate chemotherapy, v) for salvage therapy after disease progression, vi) patients who have failed multiple other standard therapies, vii) patients with disease progression after autologous stem cell transplant, viii) patients with disease progression after allogeneic stem cell transplant, and ix) as a bridging therapy prior to allogeneic stem cell transplant.

[0129] Additionally, in the human setting, B-NHL patients with diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, and mantle cell lymphoma with the following characteristics will be evaluated through a Phase 1 clinical trial: These patients are: i) patients with multidrug resistance, ii) patients unsuitable for allogeneic stem cell transplantation, iii) patients with the presence of comorbidities precluding further chemotherapy, iv) elderly patients unable to tolerate chemotherapy, v) patients for salvage therapy after disease progression and patients for whom multiple other standard therapies have failed, vi) patients with disease progression after autologous stem cell transplantation, vii) patients with disease progression after allogeneic stem cell transplantation, viii) patients for whom it is applied as a bridging therapy before allogeneic stem cell transplantation, ix) patients who exhibit escape variants or escape mutants of CD19 and / or CD20 on the surface of tumor cells, thus making current antibody therapy (anti-CD20, rituximab, anti-CD19, oretuzumab, BITE CD19 / CD2, brimatumonab) or anti-CD19 CAR therapy ineffective due to loss / downregulation of its target structure.

[0130] An additional surprising aspect of the present invention is the enhanced stability of the CAR disclosed herein: CAR polypeptides can be easily stored under appropriate conditions for extended periods of time without any loss of binding affinity.

[0131] Chimeric Antigen Receptor: CARs consist of an extracellular domain derived from an antibody and an intracellular domain comprising a signaling module derived from a T cell signaling protein. In a preferred embodiment, the extracellular domain preferably comprises variable regions from the heavy and light chains of an immunoglobulin configured as a single chain variable fragment (scFv). The scFv is preferably attached to a hinge region that provides flexibility and transmits the signal through a fixed transmembrane portion to the intracellular signaling portion. The transmembrane domain is preferably derived from CD8α or CD28. In first generation CARs, the signaling domain consists of the ζ chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domain. Second generation CARs are equipped with a single costimulatory domain derived from CD28 or 4-1BB. Third generation CARs already contain two costimulatory domains (e.g. CD28, 4-1BB, ICOS, OX40, CD3ζ). The invention preferably relates to second or third generation CARs.

[0132] In various embodiments, engineered receptors are provided that redirect the cytotoxicity of immune effector cells to B cells. These engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., BCMA) with a T cell receptor activation intracellular domain to generate chimeric proteins that exhibit specific anti-BCMA cellular immune activity. As used herein, the term "chimeric" describes the composition of different protein or DNA portions from different sources.

[0133] The CARs discussed herein contain an extracellular domain (also referred to as a binding domain or antigen binding domain) that binds to BCMA, a transmembrane domain, and an intracellular or intracellular signaling domain. The anti-BCMA antigen binding domain of the CAR binds to BCMA on the surface of the target cell, resulting in clustering of the CAR and delivery of an activating stimulus to the CAR-containing cell. A key feature of CARs is their ability to redirect the specificity of immune effector cells, thereby initiating the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of cells expressing the target antigen, independent of major histocompatibility (MHC), and the ability to target monoclonal antibodies, soluble ligands, or cell-specific co-receptors in a cell-specific manner.

[0134] In various embodiments, the CAR comprises an extracellular binding domain comprising a humanized BCMA-specific binding domain; a transmembrane domain; and one or more intracellular signaling domains. In particular embodiments, the CAR comprises an extracellular binding domain comprising a humanized anti-BCMA antigen-binding fragment; one or more spacer domains; a transmembrane domain; and one or more intracellular signaling domains.

[0135] "Extracellular antigen binding domain" or "extracellular binding domain" are used interchangeably and provide a CAR with the ability to specifically bind to the target antigen of interest, BCMA. The binding domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. Preferred is an scFv domain.

[0136] "Specific binding" should be understood as understood by those skilled in the art, and by this term the skilled artisan clearly recognizes the various experimental procedures available for investigating binding and binding specificity. Methods for determining equilibrium association or dissociation constants are known in the prior art. Although some cross-reactivity or background binding may be unavoidable in many protein-protein interactions, it does not detract from the "specificity" of the binding between the CAR and the epitope. "Specific binding" describes that the anti-BCMA antibody or antigen-binding fragment thereof (or the CAR comprising it) binds to BCMA with a binding affinity greater than background binding. The expression "towards" can also be applied when considering the term "specificity" in understanding the interaction between the antibody and the epitope.

[0137] "Antigen (Ag)" refers to a compound, composition, or substance capable of eliciting the production of antibodies or stimulating a T cell response in an animal. In particular embodiments, the target antigen is an epitope of a BCMA polypeptide. "Epitope" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous or non-contiguous amino acids that are adjacent due to the tertiary folded structure of a protein.

[0138] A "single chain Fv" or "scFv" antibody fragment comprises an antibody VH and VL domains, which are present in either orientation (e.g., VL-VH or VH-VL) in a single chain polypeptide. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR discussed herein comprises an antigen-specific binding domain, called an scFv. The scFv can be a mouse scFv, a human scFv, or a humanized scFv. The single chain antibody can be cloned to form the V region genes of a hybridoma specific for a desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human BCMA polypeptide. One non-limiting example of a variable heavy chain suitable for constructing the anti-BCMA CAR discussed herein includes the amino acid sequence shown in SEQ ID NO:11. One non-limiting example of a variable light chain suitable for constructing the anti-BCMA CAR discussed herein includes the amino acid sequence shown in SEQ ID NO:12.

[0139] Antibodies and antibody fragments: The CAR comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide. Thus, non-limiting examples of antibodies or antibody fragments of the invention include polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain fragments (scFv), single chain variable fragments (ssFv), single domain antibodies (e.g., VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments generated by Fab expression libraries, anti-idiotypic antibodies, epitope-binding fragments, or any combination thereof, provided that they retain similar binding properties as the CARs described herein, and preferably contain corresponding CDRs, or VH and VL regions as described herein. Mini-antibodies and multivalent antibodies (e.g., bivalent, trivalent, tetravalent, pentavalent antibodies) can also be used in the methods of the invention. The immunoglobulin molecules of the invention can be of any class (i.e., IgG, IgE, IgM, igD, IgA) or subclass of immunoglobulin molecules. Thus, as used herein, the term antibody also includes antibodies and antibody fragments consisting of the CAR of the present invention, either produced by modification of a whole antibody or synthesized de novo using recombinant DNA technology.

[0140] As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments thereof. When the term "antibody" is used, it can be considered to also refer to the term "antibody fragment". Known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, and epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The basic structural unit of an immunoglobulin (antibody) is known to comprise a tetramer or a dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain (about 25 kD) and one "heavy" (H) chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to the variable regions of the light and heavy chains, respectively. In some cases, antibodies, or immunogenic portions of antibodies, can be chemically conjugated to or expressed as fusion proteins with other proteins.

[0141] The CARs of the present invention are envisioned to bind to mammalian, particularly human, protein targets. The protein names used may correspond to mouse or human proteins.

[0142] The affinity of a binding domain polypeptide according to the present disclosure to a CAR protein can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay) using a labeled ligand, binding association assays, displacement assays, or using a surface plasmon resonance instrument (e.g., Biacore).

[0143] Humanized antibodies comprising one or more CDRs of or derived from an antibody of the invention can be made using any method known in the art. For example, a monoclonal antibody can be humanized using four general steps: (1) determining the nucleotide and predicted amino acid sequence of the light and heavy chain variable domains of the starting antibody, (2) designing the humanized antibody, i.e., determining which antibody framework regions should be used in the humanization process, (3) the actual humanization method / technique, and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; and 6,548,640.

[0144] The term humanized antibody means that at least part of the framework regions and possibly part of the CDR regions or other regions involved in immunoglobulin binding are derived from or adapted to human immunoglobulin sequences. Humanized, chimeric or partially humanized versions of mouse monoclonal antibodies can be produced, for example, by recombinant DNA techniques, starting from mouse and / or human genomic DNA sequences encoding H or L chains or starting from cDNAs encoding H or L chains (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibodies used in the method of the invention can be human monoclonal antibodies. Human antibodies can be obtained, for example, by using phage display techniques (WO 91 / 17271; WO 92 / 01047).

[0145] As used herein, humanized antibodies also refer to specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) forms of non-human (e.g., mouse, camel, llama, shark) antibodies that contain as little sequence derived from non-human immunoglobulin as possible.

[0146] As used herein, human antibody, human antibody fragment, humanized antibody, humanized antibody fragment refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the human antibody production techniques known in the prior art or disclosed herein. Human antibodies or fragments thereof can be selected by competitive binding experiments or otherwise to have the same epitope specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly share to a large extent the useful functional properties of mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogen. In some cases, such polyclonal antibodies can be enriched by affinity purification using amyloid fibrillar and / or nonfibrillar polypeptides or fragments thereof as affinity reagents. Monoclonal antibodies can be obtained from serum according to the techniques described in WO 99 / 60846.

[0147] Variable regions and CDRs The variable region of an antibody refers to either the variable region of the antibody light chain or the variable region of the antibody heavy chain alone, or a combination of both. Each of the heavy and light chain variable regions consists of four framework regions (FRs) connected by three complementarity determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held in close proximity to each other by the FRs and contribute to forming the antigen-binding site of the antibody with the CDRs from the other chain.

[0148] There are many techniques available for determining CDRs, including approaches based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, MD); approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). Alternative approaches include the IMGT International ImMunoGeneTics information system (Marie-Paule Lefranc). The Kabat definition is based on sequence variability and is the most commonly used method. The Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the two and is used in Oxford Molecular's AbM antibody modeling software (www.bioinf.org.uk: Andrew CR (See Dr. Martin's group.) As used herein, CDRs can refer to CDRs defined by one or more approaches, or a combination of these approaches.

[0149] In some embodiments, the invention provides an antibody or fragment thereof to be incorporated into a CAR that comprises at least one, or at least two, or at least three, or more CDRs substantially identical to at least one, or at least two, or at least three, or more CDRs of an antibody of the invention. Other embodiments include antibodies with at least two, or three, or four, or five, or six CDRs that are substantially identical to or derived from at least two, or three, or four, or five, or six CDRs of an antibody of the invention. In some embodiments, at least one, or two, or three, or four, or five, or six of the CDRs are at least about 70%, or 75%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% identical to at least one, or two, or three CDRs of an antibody of the invention. For purposes of the present invention, it is understood that binding specificity and / or overall activity will generally be retained, although the magnitude of activity may differ (may be greater or may be less) from that antibody.

[0150] Additional features of CAR In some embodiments, the CARs discussed herein can include linker residues between the various domains, which are added to provide the proper spacing and conformation of the molecule. For example, the linker includes an amino acid sequence that connects the VH and VL domains and provides a spacer function for the interaction of the two sub-binding domains, so that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody that includes the same light and heavy chain variable regions. The CARs discussed herein can include one, or two, or three, or four, or five, or more linkers. In particular embodiments, the length of the linker is about 1 to about 25 amino acids, or about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any length in between.

[0151] Exemplary linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art (e.g., Whitlow linkers). Glycine and glycine-serine polymers are relatively unstructured and may therefore serve as neutral tethers between domains of a fusion protein (e.g., a CAR as described herein).

[0152] In particular embodiments, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides", which are regions that separate the antigen binding domain from the effector cell surface to allow proper cell / cell contact, antigen binding, and activation. In some embodiments, the spacer domain is a portion of an immunoglobulin, including but not limited to, one or more heavy chain constant regions (e.g., CH2 and CH3). The spacer domain can comprise the amino acid sequence of a native immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4.

[0153] The binding domain of the CAR can in some embodiments be followed by one or more "hinge domains" that serve to position the antigen binding domain away from the surface of the effector cell to allow for proper cell / cell contact, antigen binding, and activation. The CAR can comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a natural immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative examples of hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (CD8α, CD4, CD28, PD1, CD152, CD7). The hinge region can be the wild-type hinge region from these molecules or can be altered. In another embodiment, the hinge domain comprises a PD1 hinge region, a CD152 hinge region, or a CD8α hinge region.

[0154] The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding portion with the intracellular signaling domain, and anchors the CAR to the cell membrane of the immune effector cell. The transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The transmembrane domain may be derived from the α, β, or ζ chain of the T cell receptor, or from CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD1. In one embodiment, the CAR discussed herein comprises a transmembrane domain derived from CD8α or CD28.

[0155] In a particular embodiment, the CAR discussed herein comprises an intracellular signaling domain. By "intracellular signaling domain" is meant the portion of the CAR that is involved in transmitting the message that the anti-BCMA CAR has successfully bound to the human BCMA polypeptide to the inside of the immune effector cell to induce the effector cell's function (e.g., activation, cytokine production, proliferation, cytotoxic activity, including the release of cytotoxic factors to the target cell to which the CAR is bound, and other cellular responses induced by the binding of antigen to the extracellular CAR domain). The term "effector function" means the specialized function of the immune effector cell. The effector function of T cells can be, for example, aid or activity such as cytolytic activity and secretion of cytokines. Thus, the term "intracellular signaling domain" means the portion of the protein that transmits the effector function signal and directs the cell to perform a specialized function.

[0156] The CARs discussed herein contain one or more costimulatory signaling domains that increase the efficiency and / or proliferation and / or memory formation of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule, a cell surface molecule other than an antigen receptor or an Fc receptor, that binds to an antigen and provides a second signal required for efficient activation and function of T lymphocytes.

[0157] Polypeptides The terms "peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably, unless otherwise indicated, and are used in their ordinary sense, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length and can include, for example, a full-length protein sequence or a fragment of a full-length protein, and can include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other modifications known in the art (both naturally occurring and non-naturally occurring modifications are possible).

[0158] In various embodiments, the CAR polypeptides discussed herein include a signal (or leader) sequence located at the N-terminus of the protein to co-translationally or post-translationally direct transport of the protein. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides discussed herein specifically include the CARs of the present disclosure and sequences of the CARs disclosed herein that have one or more amino acid deletions, and / or additions, and / or substitutions.

[0159] As used herein, "isolated peptide," "isolated polypeptide," and the like, refer to a peptide or polypeptide molecule that has been isolated and / or purified from an in vitro cellular environment and from association with other components of a cell, i.e., the peptide or polypeptide is not significantly associated with endogenous materials. Similarly, an "isolated cell" refers to a cell obtained from an endogenous tissue or organ and that is substantially free of extracellular matrix.

[0160] nucleic acid As used herein, "polynucleotide" or "nucleic acid molecule" refers to any of messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), and recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Polynucleotides of the invention preferably include polynucleotides or variants that are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the reference sequences described herein, and typically the variants retain at least one biological activity of the reference sequence. In various illustrative examples, some of the invention contemplates polynucleotides, including expression vectors, viral vectors, and delivery plasmids, compositions, and cells containing the same.

[0161] Polynucleotides can be prepared and / or manipulated and / or expressed using any of a variety of well-established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transcribable elements. Non-limiting examples of additional examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), PI-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phage, MI 3 phage), and animal viruses. Non-limiting examples of categories of animal viruses useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In a particular embodiment, the coding sequence of the chimeric protein disclosed herein can be ligated into such an expression vector to express the chimeric protein in mammalian cells. "Control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector (origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Dalgarno or Kozak sequence), intron, polyadenylation sequence, 5' untranslated region, 3' untranslated region) that interact with host cell proteins to effect transcription and translation. Such elements can vary in length and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, can be used.

[0162] vector In particular embodiments, a retroviral vector (e.g., a lentiviral vector) encoding a CAR is used to transduce cells (e.g., immune effector cells, such as T cells). For example, immune effector cells can be transduced with a vector encoding a CAR that comprises a humanized anti-BCMA antibody or antigen-binding fragment that binds a BCMA polypeptide and has a transmembrane domain and an intracellular signaling domain, such that the transduced cells can induce a CAR-mediated cytotoxic response.

[0163] Retroviruses are common tools for gene delivery. In a particular embodiment, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its own genomic RNA into a linear, double-stranded DNA copy and then covalently integrates the genomic DNA with the host's genome. Once integrated into the host's genome, the virus is called a "provirus." The provirus serves as a template for RNA polymerase II to direct the expression of RNA molecules that code for structural proteins and enzymes required for the production of new viral particles.

[0164] Non-limiting examples of suitable retroviruses for use in particular embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), Gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0165] As used herein, the term "lentivirus" refers to a group (or genus) of various retroviruses. Non-limiting examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In a particular embodiment, lentiviruses are used to deliver polynucleotides comprising CAR to cells.

[0166] As used herein, the term "vector" refers to a nucleic acid molecule capable of introducing or transporting another nucleic acid molecule. The introduced nucleic acid is generally linked (e.g., inserted) into the nucleic acid molecule of the vector. The vector may contain sequences directing autonomous replication in a cell or may contain sequences sufficient to allow integration with the DNA of the host cell. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and replication-defective lentiviruses.

[0167] As will be appreciated by those of skill in the art, the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a delivery plasmid) that typically contains virally derived nucleic acid elements that facilitate the introduction or integration of a nucleic acid molecule into the genome of a cell, or to a viral particle that mediates the transfer of nucleic acid. Viral particles typically contain various viral components, and sometimes host cellular components in addition to the nucleic acid.

[0168] The term viral vector can refer to a virus or viral particle capable of introducing a nucleic acid into a cell, or to the introduced nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0169] Thus, in a preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding a CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences facilitating expression of the CAR, such as a promoter, and / or an enhancer, and / or a poly-A signal, and / or one or more introns. In a preferred embodiment, the sequence encoding the CAR is flanked by transposon sequences, such that the presence of a transposase allows the integration of the coding sequence into the genome of the transfected cell.

[0170] In some embodiments, the genetically transformed cells are further transfected with a transposase that facilitates integration of the CAR coding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or protein that does not result in long-term expression of the transposase in the transgenic cells. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA that includes a cap and a poly-A tail). Any transposase system can be used in the embodiments of the invention. However, in some embodiments, the transposase is a salmonid-type Tel-like transposase (SB). For example, the transposase can be the so-called "sleeping beauty" transposase. See, e.g., U.S. Patent No. 6,489,458, which is incorporated herein by reference. In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some non-limiting examples of transposases include SB 10 transposase, SB 11 transposase, and SB 100X transposase (see, e.g., Mates et al., 2009, Nat Genet. 41(6):753-761, or U.S. Patent No. 9,228,180, which are incorporated by reference herein). For example, one method can include electroporating a cell with mRNA encoding either SB 10 transposase, SB 11 transposase, or SB 100X transposase.

[0171] Array variants: Also included within the scope of the present invention are sequence variants (e.g., sequence variants defined by % sequence identity) of the nucleic acids, and / or proteins, and / or antibodies, and / or antibody fragments, and / or CARs of the present invention that maintain similar binding properties to those of the present invention. Such variants that differ in sequence from the specific sequences provided but maintain substantially the same binding properties (e.g., target specificity) are known as functional analogs, or functionally similar. Sequence identity refers to the percentage of nucleotide or amino acid identity when sequences are aligned.

[0172] As used herein, "sequence identity" refers to the degree to which sequences match nucleotide by nucleotide or amino acid by amino acid over a comparison window.Accordingly, "sequence identity percentage" can be calculated by comparing two sequences that are optimally aligned over a comparison window, determining the number of positions in both sequences that match nucleic acid bases (e.g., A, T, G, C, I) or amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met) to obtain the number of matches, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotide and polypeptide variants that have at least about 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% sequence identity to any of the reference sequences described herein, and typically the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0173] Those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity with the nucleotide sequence of any naturally occurring gene. However, polynucleotides that differ due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions, and other changes in the sequence that correspond to the sequence identity described herein are also included in the present invention.

[0174] The scope of the present invention also includes changes in protein sequence that may occur through substitution. Substitutions, as defined herein, are changes made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the original protein. Preferably, substitutions can be made without significantly altering the function of the protein. As with additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is especially true when the change concerns "conservative" amino acid substitutions, where one amino acid is replaced with another amino acid of similar properties. Such "conserved" amino acids can be natural or artificial amino acids that can be replaced based on size, charge, polarity, conformation without significantly affecting the structure and function of the protein. Often, many amino acids can be replaced with conservative amino acids without adversely affecting the function of a protein.

[0175] In general, the following conserved amino acid groups are represented: non-polar amino acids Gly, Ala, Val, Ile, Leu; non-polar aromatic amino acids Phe, Trp, Tyr; neutral polar amino acids Ser, Thr, Cys, Gln, Asn, Met; positively charged amino acids Lys, Arg, His; and negatively charged amino acids Asp, Glu. This list is not exhaustive. For example, Ala, Gly, Ser, and sometimes Cys can substitute for each other despite belonging to different groups.

[0176] Substitutional variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, although FR changes are also considered. If such substitutions alter biological activity, more substantial changes (those marked "Example Substitutions" in the table immediately below, or those described in more detail below with reference to classes of amino acids) can be introduced and the products screened.

[0177] [Table 3]

[0178] Substantial alteration of the biological properties of the antibody can be achieved by selecting substitutions that have significantly different effects on (a) the structure of the polypeptide backbone (e.g., sheet or helical conformation) in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains.

[0179] Conservative amino acid substitutions are not limited to natural amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are omega amino acids with various chain lengths, as well as the neutral non-polar analogue cyclohexylalanine; the neutral non-polar analogues citrulline and methionine sulfoxide; the aromatic neutral analogue phenylglycine; the negatively charged analogue cysteic acid; and the positively charged analogue ornithine. As with the natural amino acids, this list is not exhaustive, but merely illustrative of the substitutions well known in the art.

[0180] Genetically modified cells and immune cells In particular embodiments, the present invention contemplates cells genetically modified to express the CAR discussed herein for use in treating B cell-related diseases. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms "genetically modified cell", "modified cell" and "redirected cell" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material in a cell to restore, correct or change the expression of a gene or to express a therapeutic polypeptide (e.g., a CAR). In particular embodiments, the CAR discussed herein is introduced and expressed in immune effector cells, redirecting their specificity to a target antigen of interest (e.g., a BCMA polypeptide).

[0181] An "immune cell" or "immune effector cell" is any cell in the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC).

[0182] Immune effector cells of the present invention can be autologous ("self") or non-autologous ("non-autologous", e.g., allogeneic, syngeneic, xenogeneic). As used herein, "autologous" refers to cells from the same subject and is a preferred embodiment of the present invention. As used herein, "allogeneic" refers to cells derived from the same species but genetically different from the comparison cell. As used herein, "syngeneic" refers to cells derived from a different subject but genetically the same as the comparison cell. As used herein, "xenogeneic" refers to cells of a different species than the comparison cell. In preferred embodiments, the cells of the present invention are autologous or allogeneic.

[0183] Examples of immune effector cells used with the CAR discussed herein include T lymphocytes. The term "T cell" or "T lymphocyte" is known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), and activated T lymphocytes. Cytokine-induced killer cells (CIK cells) are typically CD3-positive and CD56-positive non-tumor tissue-specific complex (MHC)-restricted natural killer (NK)-like T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) cell or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + These may be T cells, CD4 CD8 T cells, or any other subset of T cells. Examples of other populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.

[0184] For example, T cells modified with the CAR of the invention described herein can recognize and kill tumor cells when reintroduced into the patient after autologous cell transplantation. CIK cells are a preferred embodiment of the immune cells of the invention, as they have greater cytotoxic activity compared to other T cells.

[0185] As will be appreciated by those skilled in the art, other cells can be used as immune effector cells bearing the CARs described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursor cells of effector cells, which can be induced to differentiate into immune effector cells in vivo or in vitro.

[0186] The present invention provides a method for producing immune effector cells that express a CAR as discussed herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual to cause the immune effector cells to express one or more CARs as discussed herein. In some embodiments, the immune effector cells are genetically modified after isolation from the individual without further manipulation in vitro. Such cells can then be directly re-administered to the individual. In yet another embodiment, the immune effector cells are first activated and stimulated to expand in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express a CAR as discussed herein).

[0187] In particular embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In particular embodiments, the CAR-modified immune effector cells include T cells. T cells can be obtained from a number of sources, non-limiting examples of which include 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. In some embodiments, T cells can be obtained from a volume of blood collected from a subject using any of a number of techniques known to those skilled in the art, such as sedimentation (e.g., FICOLL™ separation), antibody-conjugated bead-based methods (e.g., MACS™ separation (Miltenyi)). In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for further processing. The cells can be washed with PBS or other suitable solutions that are calcium-free, magnesium-free, and free of most, if not all, divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known in the art, such as semi-automated flow-through centrifugation, such as the Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solutions that contain or do not contain buffers. In some embodiments, undesirable components of the apheresis sample can be removed directly from the medium in which the cells are resuspended.

[0188] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells can be further isolated by positive or negative selection techniques. One method used herein is cell sorting and / or selection through negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the surface of negatively selected cells.

[0189] PBMCs can be directly genetically modified to express CARs using the methods discussed herein. In some embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and in some embodiments, both cytotoxic and helper T lymphocytes can be classified into subpopulations of naive T cells, memory T cells, and effector T cells before or after genetic modification and / or expansion. CD8 + The cells can be obtained using standard methods. In some embodiments, the CD8 + Further classification of cells into naive, central memory, and effector cells reveals that these types of CD8 + This is done by identifying the cell surface antigens associated with each of the cells.

[0190] Immune effector cells (e.g., T cells) can be isolated and then genetically modified using known methods, or immune effector cells can be activated and expanded (or differentiated, in the case of precursor cells) in vitro and then genetically modified. In one particular embodiment, immune effector cells (e.g., T cells) are genetically modified with a chimeric antigen receptor as discussed herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after being genetically modified to express a CAR, for example, by utilizing methods described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005.

[0191] In yet another embodiment, a population of immune effector cells from a donor can be genetically modified with, for example, a mixture of one, two, three, four, five, or more different expression vectors, each vector encoding a different chimeric antigen receptor protein as discussed herein. The resulting modified immune effector cells form a mixed population of modified cells, which includes a proportion of modified cells expressing two or more different CAR proteins.

[0192] In one embodiment, the present invention provides a method for storing immune effector cells expressing genetically modified mouse CAR protein, or human CAR protein, or humanized CAR protein, and targeting BMCA protein, comprising cryopreserving the immune effector cells so that the cells remain viable when frozen. A portion of the immune effector cells expressing the CAR protein can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients affected by B cell-related diseases. The cryopreserved transformed immune effector cells can be thawed and expanded to increase the volume when needed to produce more of such cells.

[0193] Compositions and Formulations

[0194] The compositions discussed herein may include one or more of the polypeptides, polynucleotides, vectors containing same, genetically modified immune effector cells, etc. discussed herein. Compositions include, but are not limited to, pharmaceutical compositions. By "pharmaceutical composition" is meant a composition in the form of a medicament or physiologically acceptable solution for administration to a cell or animal, either alone or in combination with one or more other therapeutic modalities. It will also be understood that the compositions of the present invention may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, and a variety of other medicament active agents, if desired. There is virtually no limit to the other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to achieve the intended treatment.

[0195] The phrase "pharmaceutical acceptable" as used herein means compounds, and / or materials, and / or compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, and / or present a reasonable benefit / risk ratio.

[0196] As used herein, non-limiting examples of "pharmaceutical acceptable carriers, diluents, and excipients" include any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, and emulsifier approved by the United States Food and Drug Administration as acceptable for human and veterinary use. Non-limiting examples of pharma- ceutically acceptable carriers include sugars (e.g., lactose, glucose, sucrose); starches (e.g., corn starch, potato starch); cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, cellulose acetate); tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil); glycols (e.g., propylene glycol); polyols (e.g., glycerin, sorbitol, mannitol, polyethylene glycol); esters (e.g., ethyl oleate, ethyl laurate); agar; buffers (e.g., magnesium hydroxide, aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

[0197] In particular embodiments, the compositions of the invention comprise an amount of the CAR-expressing immune effector cells discussed herein. As used herein, the term "amount" refers to an "effective amount" or "amount effective" of the genetically modified therapeutic cells (e.g., T cells) to achieve a beneficial or desired prophylactic or therapeutic effect, including a clinical outcome.

[0198] "Prophylactically effective amount" refers to an amount of genetically modified therapeutic cells to achieve a desired prophylactic result. Because a prophylactic amount is used in a subject before the onset or at an early stage of disease, a prophylactically effective amount is typically, but not necessarily, less than a therapeutically effective amount. The term prophylactic does not necessarily mean complete inhibition or prevention of a particular medical disorder. The term prophylactic can also mean a reduction in the risk of a medical disorder occurring or worsening.

[0199] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the positive effects of the treatment outweigh any toxic or detrimental effects of the virus or transduced therapeutic cells. The expression "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When referring to a therapeutic amount, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In general, the pharmaceutical compositions comprising T cells described herein may be administered in a dosage of 10 cells per kg of body weight. 2 ~10 10 10 cells / kg body weight 5 ~10 6It can be said that the composition can be administered in a dose of 1000 cells, including any integer value within this range. The number of cells will vary depending on the end use of the composition and the type of cells contained therein. For the applications presented herein, the cells will generally be in a volume of 1 liter or less, and can be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically greater than 10 cells. 6 cells / ml, generally 10 7 > 10 cells / ml 8 Clinically significant numbers of immune cells are distributed across multiple infusions, with cumulative values ​​exceeding 10 cells / mL or more. 5 pieces, 10 6 pieces, 10 7 10 or more 8 10 or more 9 10 or more 10 10 or more 11 10 or more 12 The number of cells can be more than one. In some aspects of the invention, fewer cells can be administered, especially since all cells introduced by infusion will be redirected to a specific target antigen. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. The cells can be allogeneic, syngeneic, xenogeneic, or autologous to the patient being treated.

[0200] In general, compositions comprising cells activated and expanded as described herein can be used to treat and prevent diseases occurring in immunosuppressed individuals. In particular, compositions comprising CAR-modified T cells as discussed herein can be used to treat B-cell malignancies. The CAR-modified T cells of the present invention can be administered alone or in combination with carriers, diluents, excipients, and / or in combination with other components (e.g., IL-2 or other cytokines) or cell populations. In a particular embodiment, the pharmaceutical compositions discussed herein comprise a quantity of genetically modified T cells together with one or more pharma- ceutical or physiologically acceptable carriers, diluents, excipients.

[0201] Pharmaceutical compositions of the invention comprising a CAR-expressing immune effector cell population (e.g., T cells) can include a buffer (e.g., neutral buffered saline, phosphate buffered saline, etc.); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA and glutathione); adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the invention are preferably formulated for parenteral administration (e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration).

[0202] Liquid pharmaceutical compositions, whether in the form of a solution, suspension, or other similar form, may contain one or more of the following: a sterile diluent (e.g., water for injection, saline (preferably saline), Ringer's solution, isotonic sodium chloride), a fixed oil (e.g., synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol, and other solvents that can serve as solvents or suspending media); antibacterial agents (e.g., benzyl alcohol and methyl parabens); antioxidants (e.g., ascorbic acid and sodium hydrogen sulfate); chelating agents (e.g., ethylenediaminetetraacetic acid); buffers (e.g., acetates, citrates, phosphates); and tonicity adjusting agents (e.g., sodium chloride and dextrose). Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0203] In a particular embodiment, the compositions discussed herein comprise an effective amount of CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell composition can be applied alone or in combination with other known cancer treatments (e.g., radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc.). The compositions can also be administered in combination with antibiotics. Such therapeutic agents are accepted in the art as standard therapy for the individual disease states (e.g., individual cancers) described herein. Examples of therapeutic agents discussed include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, and other active adjunctive agents.

[0204] treatment The genetically modified immune effector cells discussed herein provide improved adoptive immunotherapy for use in the treatment of B cell-related diseases, non-limiting examples of which include immunoregulatory diseases and hematopoietic malignancies.

[0205] In particular embodiments, compositions comprising the CAR-expressing immune effector cells discussed herein are used to treat diseases associated with abnormal activity of B cells (also referred to as "medical disorders associated with the presence of pathogenic B cells").

[0206] As used herein, a "medical disorder associated with the presence of pathogenic B cells" or a "B cell malignancy" refers to a medical condition, such as a cancer, that forms in a B cell. In particular embodiments, compositions comprising the CAR-modified T cells discussed herein are used in the treatment of hematopoietic malignancies, non-limiting examples of which include B cell malignancies, e.g., multiple myeloma (MM), non-Hodgkin's lymphoma (NHL).

[0207] In another aspect of the invention, the CARs and CAR-Ts of the invention described herein are provided for use in the treatment of B cell mediated diseases, plasma cell mediated diseases, antibody mediated diseases or disorders, including but not limited to multiple myeloma (MM), chronic lymphocytic leukemia (CLL), non-secretory multiple myeloma, smoldering multiple myeloma, monoclonal globulinemia of undetermined significance (MGUS), isolated plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, cryoglobulinemia type I and II, light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid, epidermolysis bullosa acquisita; The disease may be any non-Hodgkin's lymphoma, B cell leukemia, Hodgkin's lymphoma (HL) in which BCMA is expressed, or any disease in which the patient develops antibodies that neutralize recombinant protein replacement therapy, which method comprises administering to the patient a therapeutically effective amount of a CAR or CAR-T as described herein.

[0208] Multiple myeloma is a B-cell malignancy characterized by the malignant transformation of a single clone of mature plasma cells. These plasma cells proliferate in the bone marrow and may invade adjacent bone and occasionally the blood. Variant forms of multiple myeloma include overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0209] Non-Hodgkin's lymphomas include a large group of cancers of lymphocytes (white blood cells). Non-Hodgkin's lymphomas can occur at any age and are often characterized by larger than normal lymph nodes, fever, and weight loss. Non-Hodgkin's lymphomas can also be present at extranodal sites, such as the central nervous system, mucosal tissues (lungs, small intestine, large intestine, gastrointestinal tract, etc.). There are many different types of non-Hodgkin's lymphomas. Non-Hodgkin's lymphomas can be divided, for example, into aggressive (fast growing) and indolent (slow growing). Non-Hodgkin's lymphomas can originate from B cells and T cells, but the terms "non-Hodgkin's lymphoma" and "B cell non-Hodgkin's lymphoma" are used interchangeably herein. B-cell non-Hodgkin lymphomas (NHL) include Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-cell lymphoma, and mantle cell lymphoma. Lymphomas that arise after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin lymphomas.

[0210] Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that slowly produces immature white blood cells called B lymphocytes or B cells. Cancer cells spread through the blood and bone marrow and can affect lymph nodes and other organs (e.g., liver, spleen). CLL eventually destroys the bone marrow. A different form of the disease is called small lymphocytic lymphoma, which is mostly localized in secondary lymphoid organs (e.g., lymph nodes, spleen).

[0211] In one embodiment of the invention, the CAR, or an immune cell expressing the CAR, is contemplated for use in the treatment of an autoimmune disease, preferably an autoimmune disease that is dependent on autoantibodies, and preferably an autoimmune disease that has an inflammatory component.The autoimmune disease of choice was Takayasu's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutaneous polyarteritis nodosa, hepatitis-associated arteritis, Behçet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, vasculitis of connective tissue diseases, Henoch-Schönlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, tropical leukocytoclastic vasculitis, and vasculitis of the cutaneous leukocytoclastic vasculitis. aortitis, sarcoidosis, Cogan syndrome, Wiskott-Aldrich syndrome, lepromatous arteritis, central nervous system focal vasculitis, thromboangiitis obliterans, paraneoplastic arteritis, urticaria, Degos disease, myelodysplastic syndrome, persistent erythema elevatus, hyperimmunoglobulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Morbus Chron, ulcerative colitis, autoimmune myositis, diabetes, Guillain-Barre syndrome, histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic rhinosinusitis, psoriasis, psoriatic arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis media, cryoglobulinemia, Sjogren's syndrome, lupus erythematosus (preferably systemic lupus erythematosus (SLE)), aplastic anemia, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic achalasia, bleomycin-induced pneumonitis, cytarabine-induced pneumonitis, autoimmune The autoimmune diseases are preferably selected from the group consisting of epidemic thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's disease, atrophic thyroiditis, Graves' disease, autoimmune polyendocrine syndrome, autoimmune Addison's syndrome, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiff-man syndrome, Goodpasture's syndrome, sympathetic ophthalmia, folliculitis, Sharp's syndrome, and Evans' syndrome. In particular, hay fever, periodontal disease, atherosclerosis, and rheumatoid arthritis are preferred, and SLE is most preferred.

[0212] Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease in which the body's immune system attacks healthy tissues in various parts of the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include painful and swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, fatigue, and very commonly hot flashes in the face.

[0213] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of a disease, disorder, or condition that can be treated using a gene therapy vector, cell-based therapeutic, or method disclosed elsewhere herein. In a preferred embodiment, the subject includes any animal exhibiting symptoms of a hematopoietic disease, disorder, or condition (e.g., B-cell malignancy) that can be treated using a gene therapy vector, cell-based therapeutic, or method disclosed elsewhere herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, guinea pigs), farm animals, domestic animals, or pets (e.g., cats and dogs). Non-human primates and human patients are included, with human patients being preferred. Exemplary subjects include humans with, diagnosed with, or at risk for B-cell malignancies.

[0214] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or symptoms of a disease or pathological condition, and may include even a slight decrease in one or more measurable markers of the disease or condition during treatment. Treatment may include, in some cases, the reduction or amelioration of symptoms of the disease or condition, or the slowing of the progression of the disease or condition. "Treatment" does not necessarily mean the eradication or complete cure of the disease or condition or its associated symptoms.

[0215] As used herein, "prevent" and similar terms such as "prevented," "preventing," "prophylactic," and the like refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. The terms also refer to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and its synonyms also include reducing the extent, and / or effect, and / or symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0216] In one embodiment, a method of treating a B cell related disorder in a subject in need thereof comprises administering an effective amount (e.g., a therapeutically effective amount) of a composition comprising a genetically modified immune effector cell as discussed herein. The amount and frequency of administration will depend on factors such as the condition of the patient, the type and severity of the patient's disorder, and appropriate dosages can be determined by clinical trials.

[0217] Administration of the compositions discussed herein can be performed in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, and implantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than intestinal and topical administration, typically by injection, non-limiting examples of which include intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one embodiment, the compositions discussed herein are administered by direct injection into a tumor, lymph node, or site of infection in a subject.

[0218] The embodiments and drawings disclosed herein will clarify the present invention through illustration. The drawings presented herein represent specific embodiments of the present invention and are not intended to limit the scope of the present invention. The drawings should be considered as providing further illustrations of possible and possibly preferred embodiments that provide greater technical support for one or more non-limiting embodiments. [Brief description of the drawings]

[0219] [Figure 1] Figure 1: Schematic of the preferred CAR structure. [Diagram 2] Figure 2: Schematic diagram of preferred CAR constructs IX, X, XI, XV, XVI, XVII. [Diagram 3] FIG. 3: List of preferred constructs of CAR described herein and possible combinations of different structural elements. [Figure 4] Figure 4: Sequence comparison between the mAb binding regions and the preferred humanized sequences used in the CAR of the present invention. [Diagram 5] Figure 5: GeneArt™ plasmid carrying the BCMA-CAR sequence. [Figure 6] Figure 6: Gel electrophoresis of constructs and vectors after restriction. [Figure 7] Figure 7: Confirmation of BCMA CAR expression in human T cells following retroviral transduction: CAR expression constructs IX-XII, CD19, SP6. [Figure 8] Figure 8: Culturing CAR-transduced human T cells with various target cell lines clearly demonstrates specific T cell activation by BCMA+ multiple myeloma (MM) and B-NHL cell lines. Functional in vitro co-culture and IFN-γ ELISA. [Figure 9] Figure 9: CD107a (LAMP1) staining of CAR-T cells cultured with multiple myeloma cells: Degranulating CD8+ T cells activated by antigen-specific (BCMA) stimulation detected by flow cytometry. Functional in vitro co-culture and LAMP1 detection by FACS. [Figure 10-1]Figure 10: Cytotoxicity assays reveal selective killing of BCMA-positive cell lines; virtually no killing was observed in BCMA-negative cell lines. Functional in vitro co-culture and 51Cr-release assays. [Figure 10-2] Figure 10: Cytotoxicity assays reveal selective killing of BCMA-positive cell lines; virtually no killing was observed in BCMA-negative cell lines. Functional in vitro co-culture and 51Cr-release assays. [Figure 11-1] Figure 11: BCMA and CD19 expression on the surface of various cell types assessed by functional assays. MACS-based isolation of B cells from PBMCs is also shown with anti-BCMA and anti-CD19 staining. [Figure 11-2] Figure 11: BCMA and CD19 expression on the surface of various cell types assessed by functional assays. MACS-based isolation of B cells from PBMCs is also shown with anti-BCMA and anti-CD19 staining. [Figure 11-3] Figure 11: BCMA and CD19 expression on the surface of various cell types assessed by functional assays. MACS-based isolation of B cells from PBMCs is also shown with anti-BCMA and anti-CD19 staining. [Figure 11-4] Figure 11: BCMA and CD19 expression on the surface of various cell types assessed by functional assays. MACS-based isolation of B cells from PBMCs is also shown with anti-BCMA and anti-CD19 staining. [Figure 12] Figure 12: Schematic representation of the binding interaction between the scFv of CAR and the BCMA epitope. [Figure 13] FIG. 13: Sequence alignment comparing the preferred humanized sequence of HC with J22.9-xi. [Figure 14] Figure 14: Sequence alignment comparing the preferred humanized sequence of LC with J22.9-xi. [Figure 15]Figure 15: BCMA-redirected CAR-T cells are effective against MM tumors in a xenograft NSG mouse model. (A) Implantation of MM tumors in a xenograft NSG mouse model. Mice were challenged with intraperitoneal implantation of MM.1S cells. Tumor cell growth was visualized by IVIS imaging 8 days after tumor inoculation. Imaging was extended to 300 seconds (day -1) to measure tumor burden. (B) Mice as in (A) were imaged again for 30 seconds on day -1 to track treatment efficacy and reduce bioluminescence intensity for better visualization. IVIS exposure for 30 seconds after implantation of CAR-T cells (control SP6 CAR-T cells (n=4) and BCMA CAR-T cells (n=6)) allowed a better comparison between day -1 and day 17, when intensity was at its maximum. White crosses: mice were euthanized due to disease progression and animal welfare reasons. (C) Mean bioluminescence signal intensity obtained from regions of interest covering the whole body of each mouse is plotted for each group at each time point. [Figure 16] Figure 16: BCMA-redirected CAR-T cells are effective against B-NHL tumors in a xenograft NSG mouse model. (A) Engraftment of mantle cell lymphoma in a xenograft NSG mouse model. Mice were challenged with 6x105 JeKo-1 cells intraperitoneally. Tumor cell growth was visualized by IVIS imaging on day 7 after tumor inoculation. IVIS exposure, 120 s. (B) To track the therapeutic effect and to reduce the bioluminescence intensity for better visualization, mice as in (A) were imaged again on day 0 for 30 s. IVIS exposure for 30 s after implantation of CAR-T cells (control SP6 CAR-T cells (n=7) and BCMA CAR-T cells (n=7)) allowed a better comparison between day 0 and day 16, when the intensity was at its maximum. (C) Mean bioluminescence signal intensity from regions of interest covering the whole body of each mouse is plotted for each group at each time point. EXAMPLES

[0220] The present invention is illustrated by the examples disclosed herein. The examples are presented as technical support and more detailed description of possible preferred non-limiting embodiments of the present invention. To demonstrate the function of the CAR described herein, the inventors carried out the following experiments: - CAR-transduced human T cells cultured with various target cell lines clearly demonstrated BCMA + Specific T cell activation by MM and NHL cell lines was observed; IFN-γ was detected as an effector cytokine released by T cells; - BCMA cytotoxicity assay + Selective killing of cell lines is evident; virtually no killing was observed in BCMA negative cell lines or primary cells (e.g. HUVEC (derived from endothelial cells), HEK293 (kidney), peripheral blood B cells, peripheral blood total lymphocytes, T-ALL, B-ALL, colon cancer). - CAR-T cells cultured with multiple myeloma cells were stained for CD107a and degranulated CD8+ cells activated by antigen-specific (BCMA) stimulation. + T cells were detected by flow cytometry. - In vivo experiments will involve obtaining i) functionality data, ii) off-target reactivity data, iii) T-cell memory data and iv) biosafety data of adoptively transferred CAR-T cells against B-NHL and multiple myeloma cell lines using a xenograft NSG mouse model. For B-NHL, the cytolytic potential of anti-BCMA CAR-T cells will be compared to an established anti-CD19 CAR-T cell product.

[0221] Example 1: Cloning and plasmid preparation: The CAR sequence was synthesized using GeneArt™ (Gene Synthesis Service). Restriction digestion of the CAR construct was performed using NotI and EcoRI (Figure 5). The retroviral vector MP71 was also digested with NotI and EcoRI and then dephosphorylated.

[0222] CAR and vector were separated using gel electrophoresis (Figure 6) and the fragment was purified. The CAR construct was then ligated to the vector (50 ng) in a 3:1 ratio. The ligation mixture was transformed into MACH-1 (Figure 3). Restriction digestion was performed and the Mini-Preparation was sequenced. The construct was then transformed again into MACH-1. A Maxi-Preparation of the MP71-BCMA-CAR plasmid was made.

[0223] MP71 is a positive-stranded RNA virus. Reverse transcriptase converts the retroviral RNA-genome into a DNA copy. The DNA integrates into the target genome at random locations as a provirus. The virus is stably reproduced as a provirus through cell division.

[0224] Example 2: Transfection and transduction: Day 0: For virus production, HekT (293T) cells or GalV cells were seeded into 6-well plates.

[0225] Day 1: Transient 3-plasmid transfection for virus production (calcium phosphate transfection). Standard protocols were followed using 18 μg of DNA per well in 250 μl CaCl2 and 150 μl H2O. Cells were incubated at 37°C for 6 hours, medium was changed, and incubated at 37°C for an additional 48 hours.

[0226] Coat 24-well non-tissue culture plates with anti-huCD3 and anti-huCD28 antibodies: Prepare anti-CD3 / anti-CD28 antibody solutions in 0.5 ml PBS per well (5 μg / ml anti-CD3 antibody, 1 μg / ml anti-CD28 antibody). Incubate each well with 0.5 ml of antibody solution for 2 hours at 37° C., replace with sterile 2% BSA solution (in water) and incubate for 30 minutes (37° C.). Remove the BSA solution and wash the wells with 2 ml PBS.

[0227] Purification of PBMCs from 40 ml of blood (approximately 2.5 × 10 7 PBMCs): 12.5 ml of Ficoll-Gradient medium is prepared in 2 x 50 ml Falcon-Tubes, blood is diluted to 45 ml with RPMI (+100 IU / ml penicillin, streptomycin), mixed, covered with 22.5 ml of blood-medium mixture and centrifuged (20 min, 20°C, 1800 rpm, RZB *648, G 17.9). 15 ml of the upper phase is discarded. The remainder of the upper phase together with the milky PBMC-containing intermediate phase is transferred to a new 50 ml Falcon-Tube, RPMI (+100 IU / ml penicillin, streptomycin) is added to 45 ml and centrifuged. Resuspend the pellet in 45 ml RPMI (+100 IU / ml penicillin, streptomycin), centrifuge, pool the pellet in 10–20 ml T cell medium, stain one sample with trypan blue, count the cells, and plate 1–1.5 × 10 cells onto wells coated with anti-CD3 and anti-CD28. 6 The remaining PBMCs are centrifuged, resuspended in freezing medium, and stored in CryoTubes at -80°C.

[0228] Day 3: Transduction of PBLs Viral supernatant is removed from Hekt or GalV cells and filtered (0.45 μm filter). Stimulated PBMCs are treated with 1.5 ml of viral supernatant.

[0229] Day 4: Transduction of PBLs Filter the remaining viral supernatant (4°C) and the second supernatant (0.45 μl) from Hekt or GalV cells. Recover 1 ml to 1.5 ml of supernatant from PBLs. Treat stimulated PBMCs with 1 ml to 1.5 ml of viral supernatant and centrifuge in CD3 / CD28 coated wells (90 min, 32°C, 2000 rpm). Final concentrations are 100 IU / ml IL-2 (400 U / μl to 1 μl) or 10 ng / μl IL-7 and 10 ng / μl IL-15 plus 4 μg / ml (8 μl) protamine sulfate. Centrifuge for 90 min at 32°C, 2000 rpm. Days 7-13: Culture PBL and T cell media and treat with fresh IL-2 or IL-7 / IL-15. Day 13: End of T cell stimulation. PBL cultures from cell culture flasks are washed, centrifuged and the pellets are resuspended in T cell medium (+10 IU / ml IL-2). Day 15: Functional assays

[0230] Example 3: Functional in vitro testing of anti-BCMA CAR-T cells I. Confirmation of BCMA CAR expression on the surface of human T cells following retroviral transduction

[0231] We obtained evidence that CAR receptors are folded and trafficked in the context of human T cells; and we assessed the functionality of a retroviral transduction protocol.

[0232] Human peripheral blood leukocytes were purified through Ficoll-Gradient. Cells were cultured, stimulated, and retrovirally transduced as described above. Following transduction, cells were further cultured in IL-2- or IL-7 / IL-15-containing medium and then analyzed for BCMA-CAR expression.

[0233] Transduction rates and viability were assessed by flow cytometry (FACS) analysis. To detect BCMA-CAR expression, cells were stained with anti-human Ig antibodies that selectively recognize the human IgG1 or IgG4 compartments within the spacer region of the CAR construct. CD3 / CD8 / CD4 T cells were stained simultaneously. See Figure 7 for results.

[0234] II. CAR-transduced human T cells cultured with various target cell lines clearly demonstrate BCMA + Specific T cell activation by multiple myeloma (MM) and B-NHL cell lines is revealed

[0235] The findings showed that IFN-γ was an effector cytokine released by T cells.

[0236] Generate retrovirally transduced human T cells as detailed previously; use all BCMA CAR receptor variants (IX-XVII), SP6 negative control CAR, CD19 CAR, UT = non-transduced T cells. Use the following human cell lines as target cells in co-culture:

[0237] [Table 4]

[0238] Retrovirally transduced T cells are co-cultured in the presence of the listed cell lines or primary cells at a 1:1 ratio for 18-20 hours. After that time, cell-free medium supernatants are harvested; the most release is induced by PMA / ionomycin stimulation of effector T cells; the least release is by T cells alone. IFN-γ release into the supernatant is examined by ELISA. See Figure 8 for results.

[0239] III. CD107a (LAMP1) staining of CAR-T cells cultured with multiple myeloma cells: Degranulated CD8 activated by antigen-specific (BCMA) stimulation + T cells detected by flow cytometry Generate retrovirally transduced human T cells as previously detailed; use BCMA CAR receptor variants (IX-XI), SP6 negative control CAR.

[0240] Retrovirally transduced T cells are co-cultured in the presence of the listed cell lines at a 1:1 ratio for 18 hours.

[0241] Anti-CD107a (LAMP1) antibodies are added to the cell culture medium after overnight incubation; the antibodies bind to T cells continuously as secretory lysosomes fuse with the plasma membrane and release enzymes in their endoplasmic reticulum. These endoplasmic reticulum contain cytolytic mediators (e.g., granzymes and perforin). The next day, T cells are stained with anti-CD8 and / or anti-CD3.

[0242] Flow cytometric analysis: greater CD107a reactivity, shown as mean fluorescence intensity (MFI), indicates stronger T cell activation. It can be confirmed that T cell activation is antigen dependent. See Figure 9 for results.

[0243] IV. Cytotoxicity assays reveal selective killing of BCMA-positive cell lines; virtually no killing of BCMA-negative cell lines 51 A Cr release assay is used to quantitate the activity of cytotoxic T lymphocytes and measure cytolysis of target cells.

[0244] Generate retrovirally transduced human T cells as previously detailed; using BCMA CAR receptor variants (IX-XI), SP6 negative control CAR; and CD19 CAR as a control.

[0245] Target cells 51 Cr. CAR-T cells are then co-cultured with the labeled target cells for 4 hours. The effector to target ratio is titrated. E:T 80:1 40:1 20:1 10:1 5:1 2.5:1

[0246] The supernatant of the cell-free cell culture is harvested. The supernatant is transferred to a LUMA-scintillation plate and the released 51 Cr is measured in a γ-scintillation counter. Highest release: target cells lysed by Triton X-100 permeabilizing agent. Lowest release: target cells only. See FIG. 10 for results.

[0247] Additionally, Figure 11 shows the amount of BCMA and CD19 expressed on the surface of each of the various cells evaluated for cytotoxicity. Figure 12 shows a schematic representation of the binding interaction between the scFv of the CAR and the BCMA epitope.

[0248] Example 4: In vivo experiments using a xenograft NSG mouse model to evaluate adoptively transferred CAR-T cells versus B-NHL and multiple myeloma cell lines In vivo experiments using xenografts in NSG mice: 1) To demonstrate that CAR-T cells with different anti-BCMA variants have effector activity under in situ conditions, multiple myeloma cells with different BCMA antigen densities were intravenously injected into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1 Wjl The multiple myeloma cell lines that can be used are RPMI-8226, which has low BCMA density; MM1S, which has intermediate BCMA density; and NCI-H229, which has high BCMA density.

[0249] 2) To confirm the reactivity of anti-BCMA CAR-T cells against B-NHL cell lines in situ, NSG mice were intravenously injected with luciferase-transduced cell lines (e.g., SU-DHL4 (DLBCL), JEKO-1 (mantle cell lymphoma), JVM3 (CLL), MEC1 (CLL), DOHH-2 (FL)).

[0250] BCMA CAR-T cells mediate in vivo antitumor activity in mouse models of multiple myeloma (MM) and B-cell non-Hodgkin lymphoma (B-NHL): To prove the concept that the robust in vitro activity of BCMA CAR-engineered T cells translates into effective antitumor activity in vivo, we used NOD.Cg-Prkdc scid Il2rg tm1 WjlCohorts of / SzJ (NSG) mice were inoculated intravenously with the human MM.1S cell line (Figure 15) or the B-NHL cell line JeKo-1 (Mantle Cell Lymphoma) (Figure 16) and transduced with the luciferase gene in tandem with GFP. NSG mice do not develop T, B or NK cells, making them well suited for tolerance and growth of xenografted human cells. No "graft versus host" (GvHD) reactions (xenoreactivity) were observed within the experimental period shown here (data not shown). Tumor growth was monitored by IVIS imaging and luciferin was injected after 7–8 days. CAR-T cells were injected intravenously one day after tumor growth was confirmed (=day 0). For functional in vivo experiments, CAR construct IX (B IX) was used. A total of 6–7 × 10 CAR-T cells were obtained per mouse. 6 The mean transduction rate of T cells within this population was 40-60%, never exceeding 100 cells. Transduction rates of SP6 and BCMA per donor were consistent within ±10%. In the two experiments shown, transduced, effective CAR-T cells were obtained from 3 × 10 6 Control mice received SP6 CAR-T cells.

[0251] In the MM1.S experiment (Figure 15), 3 x 10 transduced CAR-T cells were 6 (As mentioned above, the total is 6-7 x 10 6 100 MM cells were transplanted into 100 mice (100 MM cells) and the observation period was extended to day 17. Virtually all mice treated with SP6 CARs, but not those treated with BCMA CARs, either progressed in MM disease (disease progression is characterized by strong fluorescent signals in the spinal cord, pelvis, and hind limbs) or were euthanized due to disease progression in accordance with the animal protection law (Federal State of Berlin). We conclude that already with this relatively low number of CAR-T cells, BCMA CAR-T cells have anti-myeloma activity (Figures 15A-C).

[0252] Due to their high affinity and avidity, anti-BCMA CAR-T cells can recognize even mature B-cell NHLs with low BCMA expression, thus enabling T cell activation and tumor cell killing. Such mature B-NHLs include several stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia (see Figure 11, Figure 10, Figure 9, Figure 8). To prove that BCMA is suitable as a target structure in B-NHL, transduced CAR-T cells (total: 6-7 × 10 6 We transplanted 1000 cells (1000 BCMA CAR-T cells) into NSG mice that had been previously challenged with the mantle lymphoma cell line JeKo-1. Virtually all mice treated with the SP6 CAR progressed lymphoma (disease progression marked by strong fluorescent signals in the liver, thoracic organs, hindlimb bone marrow, and spleen), whereas the BCMA CAR-treated group apparently did not. Accordingly, we present the first preclinical in vivo evidence that BCMA CAR-T cells have antitumor activity that extends beyond multiple myeloma to B-NHL lymphoma (Figures S16A-S16C).

[0253] Example 5: Measuring the surface density of BCMA molecules Due to their high affinity and avidity, anti-BCMA CAR-T cells can recognize even mature B-cell NHLs with low BCMA expression, leading to T cell activation and tumor cell killing, including several stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

[0254] To quantify the surface density of BCMA molecules A PE phycotoerythrin fluorescent detection kit (also called BD Quantibrite assay (BD Biosciences)) was applied. The number of PE molecules per cell can be converted to the number of antibodies per cell, which is a quantitative estimate of the number of antigens per cell. A flow cytometry detection method was applied.

[0255] Using this method, we found that the multiple myeloma cell line NCI-H929 has a relative surface BCMA antigen density of 12555, the multiple myeloma cell line OPM-2 has 3443 BCMA molecules, and the multiple myeloma cell line MM.1S has a relative value of 3181.

[0256] The BCMA antigen density of the mentioned B-NHL cell lines is For NCI-H929 , DOHH-2: 1 / 20, JeKo-1: 1 / 250, and MEC-1: 1 / 34.

Claims

1. i. an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a B-cell maturation antigen (BCMA) polypeptide; ii. a transmembrane domain; iii. Intracellular domain and an isolated chimeric antigen receptor (CAR) polypeptide that binds to an epitope comprising one or more amino acids from residues 13 to 32 of the N-terminus of human BCMA.

2. The extracellular antigen-binding domain comprises: - a heavy chain complementarity determining region 1 (H-CDR1) that has at least 80% sequence identity with SEQ ID NO: 1 (GFTFSRYW); - a heavy chain complementarity determining region 2 (H-CDR2) that has at least 80% sequence identity with SEQ ID NO: 2 (INPSSSTI); - a heavy chain complementarity determining region 3 (H-CDR3) that has at least 80% sequence identity with SEQ ID NO: 3 (ASLYYDYGDAYDY); and a variable heavy chain (VH) comprising - a light chain complementarity determining region 1 (L-CDR1) that has at least 80% sequence identity with SEQ ID NO: 4 (QSVESN); - a light chain complementarity determining region 2 (L-CDR2) that has at least 80% sequence identity with SEQ ID NO:5 (SAS); - a light chain complementarity determining region 3 (L-CDR3) that has at least 80% sequence identity with SEQ ID NO:6 (QQYNNYPLT); 2. The isolated chimeric antigen receptor (CAR) polypeptide of claim 1, comprising a variable light chain (VL) comprising:

3. - GFTFSRYW (H-CDR1; SEQ ID NO: 1), -INPX 2 X 3 STI (H-CDR2; SEQ ID NO: 7) (where X 2 X 3 are SS, NS, TS, GS, KS, RS, SD, SN, and DE) and - ASLYX 4 DYGDAX 5 DY (H-CDR3; SEQ ID NO: 8) (wherein X 4 is Y, L, A, V, F, I, W, and / or X 5 are Y, L, F, I, V, A, and C) A VH domain comprising the CDR sequence: - QSVX 1 X 2 N (L-CDR1; SEQ ID NO: 9) (where X 1 X 2 are ES, SS, TS, QS, HS, and DH) and - SAS (L-CDR2; SEQ ID NO: 5), - QQYNNYPLTFG (L-CDR3; SEQ ID NO: 10) 3. The isolated chimeric antigen receptor (CAR) polypeptide of claim 1 or 2, comprising a VL domain comprising the CDR sequences:

4. The following sequence: i. H-CDR1: GFTFSRYW (SEQ ID NO: 1); ii. H-CDR2: INPSSSTI (SEQ ID NO: 2); iii. H-CDR3: ASLYYDYGDAYDY (SEQ ID NO: 3); iv. L-CDR1: QSVESN (SEQ ID NO: 4); v. L-CDR2: SAS (SEQ ID NO:5); vi. L-CDR3: QQYNNYPLT (SEQ ID NO: 6) 4. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 3, comprising:

5. SEQ ID NO:11 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWV a VH domain having at least 80% sequence identity with the VH domain of the GEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS; SEQ ID NO:12 (EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIY 5. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 4, comprising a VL domain that has at least 80% sequence identity with (SASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK).

6. 6. The isolated chimeric antigen receptor (CAR) polypeptide of claim 5, comprising at least W36, E50, L99, Y100, Y101, A106 of SEQ ID NO:11 and at least S31, A34, S50, L53, Q89, Y91, Y94, L96 of SEQ ID NO:

12.

7. 7. The isolated chimeric antigen receptor (CAR) polypeptide of claim 5 or 6, comprising at least the CDR sequences of SEQ ID NOs: 1 to 6.

8. 8. An isolated chimeric antigen receptor (CAR) polypeptide according to one or more of claims 5 to 7, comprising a VH domain as set forth in SEQ ID NO: 11 and a VL domain as set forth in SEQ ID NO:

12.

9. 9. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 8, wherein the CAR, when expressed in a genetically modified immune cell, preferably a T lymphocyte, induces cytotoxic activity against non-Hodgkin's lymphoma (B-NHL) by binding to BCMA on the surface of the B-NHL through the CAR and activating the immune cell.

10. 10. The isolated chimeric antigen receptor (CAR) polypeptide of claim 9, wherein the B-NHL is a JeKo-1 cell line, and / or a DOHH-2 cell line, and / or a SU-DHL4 cell line, and / or a JVM-3 cell line, and / or a MEC-1 cell line.

11. 11. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 10, wherein the extracellular antigen binding domain comprises a linker polypeptide located between the VH domain and the VL domain.

12. 12. The isolated chimeric antigen receptor (CAR) polypeptide of claim 11, wherein the linker is selected from a Whitlow linker (SEQ ID NO: 13; GSTSGSGKPGSGEGSTKG), a Gly-Ser linker (SEQ ID NO: 14; SSGGGGSGGGGSGGGGS), or a linker that has at least 80% sequence identity with SEQ ID NO: 13 or 14.

13. a spacer peptide located between the extracellular antigen-binding domain and the transmembrane domain, wherein the selection of the spacer comprises: a. IgG1-CD28 spacer (SEQ ID NO: 15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPK DTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK) b. IgG1Δ-4-1BB spacer (SEQ ID NO: 16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFP PKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK) c. IgG4 (Hi-CH2-CH3) spacer (SEQ ID NO: 17; ESKYGPPCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK) d. IgG4 (Hi-CH3) spacer (SEQ ID NO: 18; ESKYGPPCPPCPGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK) e. IgG4 (Hi) spacer (SEQ ID NO: 19; ESKYGPPCPPCP) f. A spacer having a sequence that is at least 80% identical to any one of SEQ ID NOs: 15 to 19.

13. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 12, comprising:

14. The selection of the transmembrane domain is the CD8α domain (SEQ ID NO: 20; LSLVITLYC), a CD28 domain (SEQ ID NO: 21; FWVLVVVGGVLACYSLLVTVAFIIFWV), and a transmembrane domain that has at least 80% sequence identity with SEQ ID NO: 20 or 21.

15. 15. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 14, wherein the intracellular domain comprises a costimulatory domain selected from a 4-1BB costimulatory domain (SEQ ID NO: 22; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), a CD28 costimulatory domain (SEQ ID NO: 23; RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS), a costimulatory domain that has at least 80% sequence identity to SEQ ID NO: 22 or 23.

16. and a signaling domain, the signaling domain being the CD3ζ (CD28 or 4-1BB) signaling domain (SEQ ID NO: 24; LRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or a signaling domain having at least 80% sequence identity to SEQ ID NO:

24.

17. 17. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 16, comprising a tandem costimulatory domain comprising a 4-1BB costimulatory domain (SEQ ID NO: 22), a CD28 costimulatory domain (SEQ ID NO: 23), and a CD3 zeta signaling / activation domain (SEQ ID NO: 24).

18. a) - a nucleotide sequence encoding an isolated chimeric antigen receptor (CAR) polypeptide according to any one of claims 1 to 17, and / or - a nucleotide sequence comprising the sequences set forth in SEQ ID NO: 66 and / or 67, or SEQ ID NO: 86 to 94 a nucleic acid molecule comprising b) a nucleic acid molecule complementary to the nucleotide sequence of a); c) a nucleic acid molecule comprising a nucleotide sequence sufficiently identical in sequence to be functionally similar / equivalent to the nucleotide sequence set forth in a) or b), preferably at least 80% identical to the nucleotide sequence set forth in a) or b); d) a nucleic acid molecule which, as a consequence of the degeneracy of the genetic code, results in a nucleotide sequence as set forth in a) to c); e) Nucleic acid molecules according to a) to d) which have been modified by deletions, and / or additions, and / or substitutions, and / or rearrangements, and / or inversions, and / or insertions, but which are functionally similar / equivalent to the nucleotide sequences according to a) to d). An isolated nucleic acid molecule, preferably in the form of a vector (eg, a viral vector), selected from the group consisting of:

19. A genetically modified immune cell comprising the nucleic acid molecule or vector of claim 18 and / or expressing a CAR of any one of claims 1 to 17.

20. 20. The genetically modified immune cell of claim 19, selected from the group consisting of T lymphocytes and NK cells.

21. 21. The genetically modified immune cell of claim 20, wherein the T lymphocyte is a cytotoxic T lymphocyte.

22. An immune cell according to any one of claims 19 to 21 for use in the treatment of a medical disorder associated with the presence of pathogenic B cells.

23. The immune cell of claim 22 for use as a medicine, wherein the medical disorder associated with the presence of pathogenic B cells is a plasma cell disease, and / or a mature B cell disease, and / or a memory B cell disease.

24. 23. The genetically modified immune cell of claim 22 for use as a medicine, wherein the medical disorder is multiple myeloma.

25. 23. The immune cell of claim 22, for use as a medicament, wherein the medical disorder is non-Hodgkin's lymphoma.

26. 23. The immune cell of claim 22 for use as a medicine, wherein the medical disorder is an autoantibody-dependent autoimmune disease.

27. 27. The immune cell of claim 26, for use as a medicament, wherein the medical disorder is systemic lupus erythematosus (SLE) or rheumatoid arthritis.

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